Encapsulation methods and particles

Abstract

Provided herein are devices for producing aqueous core polymer shell particles described herein. Also provided herein are methods of making such particles, as well as the particles themselves. These particles are useful in medicine, especially in the vaccine context.

Description

Technical Field

[0001] The present disclosure relates to the preparation of particles comprising an aqueous core including a pharmaceutical agent encapsulated by a polymer shell. Background Art

[0002] The basis of drug therapy is to ensure that the administered agent reaches the appropriate part of the body in a manner that exerts its therapeutic effect. Although for some treatments, such as single-dose vaccinations, it is necessary to release the active agent into the body quickly (for example, in order to exert clinical benefits as quickly as possible), this is not always desirable. In some cases, delayed or sustained release formulations can be used in addition to or instead of immediate release formulations.

[0003] Delayed release of a drug can be achieved by a variety of means, depending on the therapeutic application.

[0004] Certain agents can be beneficially delivered via controlled release devices, such as infusion pumps, which can be programmed to administer the therapeutic agent according to a predetermined regimen. Such pumps range from large fixed-position devices to smaller wearable devices. However, they are physically invasive, expensive, and require careful control to ensure correct drug dosing.

[0005] Other approaches rely on formulation chemistry to provide the drug in a form suitable for controlled release therapy. A variety of approaches have been considered.

[0006] For agents that are preferably systemically administered (e.g., by intravenous injection), droplet and microencapsulation techniques have been widely proposed. One example relates to the use of liposomes. Liposomes are spherical vesicles with at least one layer of lipid (e.g., phospholipid) layer surrounding an aqueous core. Delivery can be achieved by fusing liposomes to cell membranes or by diffusion. However, although liposomes show some promise, there are still some problems. Since the lipids used to form liposomes are generally prone to degradation due to oxidation and hydrolysis, it may be challenging to ensure sufficient stability of liposomes. Physical damage to the fragile liposome structure can also lead to uncontrolled drug release. In addition, once administered, the reticuloendothelial system will generally quickly remove liposomes from the body. In addition, the cost of the lipids used for their production may be high, and the complexity of forming a homogenous sample suitable for drug delivery requires specialized equipment, which further increases the cost. Therefore, alternative and / or improved methods are needed.

[0007] A kind of alternative strategy proposed is to form droplets in which the medicament is encapsulated in a polymer. The common method adopted in this area relates to dissolving the active agent and the polymer in a suitable solvent and forming a single emulsion in an aqueous phase for delivery. The extraction of the solvent produces a porous matrix including the active agent, which is released over time along with the porous nature of (for example) the matrix and the degradation of the polymer in vivo. However, although these methods usually produce sustained release characteristics, the controlled delayed release of the agent is more challenging: in these matrix-based methods, the release of the agent usually occurs immediately after administration, and its release characteristics are determined by the matrix gradually decomposing over time. Therefore, these methods are not suitable for applications requiring delayed release characteristics, wherein the agent is only released after a period of time, rather than immediately after administration.

[0008] One situation in which a delayed (rather than sustained) release formulation is desired is vaccination. Vaccines have been developed for some of the most prevalent diseases worldwide, such as diphtheria, tetanus, pertussis, influenza and measles, helping to save millions of lives each year.

[0009] Many vaccines require multiple doses to induce durable and robust protective immunity. For example, multiple doses of a vaccine can be administered with controlled time between doses to enhance the adaptive immune response. Repeated administration can be referred to as a prime / boost regimen, in which the first administration, for example, activates naive cells. T and B cells prime the immune system by proliferating and differentiating into memory cells. The second administration may "boost" the effect of memory cells by stimulating and activating new naive cells again. Prime / boost vaccination regimens include homologous prime / boost strategies in which the same active agent is administered during the prime and boost phases, and heterologous prime / boost strategies in which different active agents are administered during the prime and boost phases.

[0010] Regardless of the exact agent being administered, the prime / boost vaccination strategy relies on multiple doses of the therapeutic agent or each therapeutic agent. This is disadvantageous compared to a single administration for several reasons. Due to the need for multiple doses, patient compliance may be reduced. Due to the need for multiple injections, the cost to medical staff may increase. The cost of distributing and storing multiple doses of vaccine may be higher than the cost of handling a single dose of vaccine. In addition, any deviation from the optimal time frame between the prime and booster administrations (e.g., due to logistical difficulties in arranging for patients to visit a medical facility at the right time) may result in reduced immunogenicity.

[0011] Therefore, new technologies for delivering vaccines are urgently needed, including the delivery of multiple doses that can be used, for example, in prime / boost vaccination regimens.

[0012] The inventors seek to develop delayed-release pharmaceutical formulations that can overcome the problems of current vaccines and related therapeutic delivery systems. The inventors have recognized that formulation methods for accurately controlling drug release will have a wide range of applications, including (but not limited to) the field of vaccination. For example, controlled delayed-release formulation technology can be used in conjunction with conventional formulations of vaccines to provide both a primary immunization dose and a booster dose in a single administration. Using such formulations can reduce costs, simplify storage, improve patient compliance, and ensure an accurate time range between a primary immunization dose and a booster dose. The disclosed devices and methods address some or all of these limitations. Summary of the invention

[0013] According to a first aspect of the present invention, a method for preparing particles is provided, the particles comprising an aqueous core encapsulated by a polymer shell, the method using a flow chip having a channel including a main channel formed in the flow chip, the method comprising: allowing a solvent phase to flow through a transmission section of the main channel, the solvent phase entraining aqueous phase droplets, wherein the aqueous phase comprises an agent and an aqueous solvent, and the solvent phase comprises a polymer and a non-aqueous solvent; allowing an extraction phase to flow through an extraction phase inlet channel in the flow chip, the extraction phase comprising an extraction solvent, wherein the extraction phase inlet channel is arranged on opposite sides of the main channel, and the transmission section and the extraction phase inlet channel lead to an extraction phase intersection section of the main channel, thereby forming solvent phase droplets encapsulating aqueous phase droplets in the extraction phase; allowing the extraction phase entrained with solvent phase droplets to flow through an extraction section of the main channel extending from the extraction phase intersection section; and, extracting the non-aqueous solvent of the aqueous phase, so as to form particles having an aqueous core formed by aqueous phase droplets and a shell formed by a polymer, wherein the main channel increases in height at or downstream of the position where the transmission section leads to the extraction phase intersection section.

[0014] Using this method, particles comprising an aqueous core encapsulated by a polymer shell can be prepared that are robust and uniform and have controllable core-shell properties.

[0015] As described in more detail herein, the particles made by the method generally include a medicament in an aqueous phase; the aqueous phase includes an aqueous solvent, surrounded by a homogeneous and uniform solid shell including a biocompatible polymer. The particles themselves are generally stable, spherical, and uniform in morphology and structure. Because their physicochemical properties can be controlled as described herein, they are ideal for delivering drugs in many different situations, including those of vaccine delivery. For example, the particles can be used to deliver discrete "boost" doses of medicaments in a primary / boost vaccination strategy.

[0016] Although the polymer particles including the medicament have been disclosed in the art, the inventors have found that these particles cannot generally provide the controlled delayed release of the medicament. Specifically, although many particles are inaccurately described in the literature as having a core surrounded by a complete shell, the fact is that such particles generally include a complex matrix of a polymer in which the agent is dispersed. Therefore, as described above, the release of the agent from the polymer is generally not discrete, and on the contrary, over time, the active agent is gradually dispersed uncontrolled from the polymer. In addition, the particles produced according to the previously known methods are generally uneven in size and thickness. In practice, they are generally not suitable for effectively delivering the medicament because the medicament is usually incorporated into the polymer matrix and the degradation of the medicament is usually caused. For example, the polymer matrix itself can cause degradation in some cases, such as by the functional group reaction of the medicament with the polymer. Alternatively or additionally, in the matrix formation and subsequent particle production process, the medicament is exposed to harsh conditions (e.g., contact with an organic solvent and / or a low pH solution) and often causes the degradation of the medicament, such as by losing the conformational structure (especially the medicament based on protein); chemical degradation; and / or polymerization. In addition, the encapsulation efficiency (i.e., drug loading) of the medicament is poor because most of the internal volume of each particle is composed of inactive polymer. The heterogeneity of the population also hinders quality control. Such prior art particles are not useful in applications where it is necessary to control the discrete release of an active agent, such as in primary / boost vaccines.

[0017] In contrast, the inventor has found that the disclosed method produces real core-shell microparticles, which have a complete shell around the aqueous core. Particles allow the core to be verifiably controlled and discrete from the shell, and are therefore significantly different from those particles that only allow the release of medicaments from the matrix gradually and / or uncontrolled in the art. In addition, particles are uniform in size and thickness. It is ideally suitable for effectively delivering medicaments, and reagents can not be degraded, particularly because any solvent and / or polymer involved in the production and storage of medicament activity can be separated. The encapsulation efficiency (i.e., drug loading) of medicament is also very high. Due to the homogenization of the colony, quality control has also been improved.

[0018] By arranging the main channel to increase in height at or downstream of the transport section leading to the extraction phase intersection section, it is achieved that stable and homogeneous particles with controllable core-shell properties are advantageously prepared. This reduces the compression of the droplets in the main channel, thereby providing stable particles with good drug loading and a high level of monodispersity of the aqueous core.

[0019] Preferably, the main channel may have an increase in height at the location where the transport section leads to the extraction phase intersection section. It has been found that such a location of increased height produces particles with particularly advantageous properties.

[0020] Alternatively, the main channel may have an increase in height downstream of the location where the transport section opens into the extraction phase junction section and upstream of the end of the nozzle section. It has been found that such a location of increased height produces particles with advantageous properties.

[0021] The method may further include forming aqueous phase droplets in the solvent phase by: allowing the aqueous phase to flow through an aqueous phase inlet section of a main channel; and allowing the solvent phase to flow through a solvent phase inlet channel within a flow chip, wherein the solvent phase inlet channel is disposed on an opposite side of the main channel, and the aqueous phase inlet section and the solvent phase inlet channel lead to a solvent phase intersection section of the main channel, so that aqueous phase droplets are formed in the aqueous phase, and the transport section extends from the solvent phase intersection section.

[0022] According to a second aspect of the present invention, there is provided an aqueous core-polymer shell particle comprising:

[0023] An aqueous core comprising a pharmaceutical agent; and

[0024] A homogeneous solid biodegradable polymer shell encapsulating an aqueous core.

[0025] The aqueous core includes the agent in an aqueous phase; the aqueous phase includes an aqueous solvent and is surrounded by a homogeneous and uniform solid shell including a biocompatible polymer.

[0026] These particles can be prepared using the methods disclosed herein.

[0027] The particles are generally stable and spherical, and are uniform in morphology and structure. Because their physicochemical properties can be controlled as described herein, they are ideal for delivering drugs in many different situations, including vaccine delivery. For example, the particles can be used to deliver discrete "boost" doses of a medicament in a prime / boost vaccination strategy.

[0028] The particles can be present in the form of a pharmaceutical composition. The pharmaceutical composition includes the particle (or multiple particles) and one or more pharmaceutically acceptable excipients, diluents or adjuvants. These components will be described in more detail herein.

[0029] In addition to the particles provided herein, the disclosed pharmaceutical compositions may also include one or more additional pharmaceutical agents.Such compositions may be particularly useful as vaccines, for example as prime / boost vaccine compositions.

[0030] According to a third aspect of the present invention, a flow chip for preparing particles is provided, the particles comprising an aqueous core encapsulated by a polymer shell, the flow chip having channels formed in the flow chip, the channels comprising a main channel and an extraction phase inlet channel arranged on opposite sides of the main channel, wherein the main channel comprises: a transmission section; an extraction phase intersection section, wherein the transmission section and the extraction phase inlet channel lead to the extraction phase intersection section; and, an extraction section extending from the extraction phase intersection section, wherein the main channel increases in height at the position where the transmission section leads to the extraction phase intersection section or at its downstream.

[0031] Such a flow chip may be used in a method according to the first aspect of the invention.

[0032] Preferably, the flow chip can be further configured so that the channel further includes a solvent phase inlet channel on the opposite side of the main channel, and the main channel further includes: an aqueous phase inlet section; and a solvent phase intersection section, and the aqueous phase inlet section and the solvent phase inlet channel lead to the solvent phase intersection section, and the transmission section extends from the solvent phase intersection section. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a plan view of a flow chip used to prepare particles;

[0034] Figure 2a and Figure 2b are aligned plan views and oblique cross-sectional views, respectively, of two alternatively configured flow chips, wherein the cross-sections are taken along the main channels;

[0035] Figure 3a to Figure 3c are aligned plan and oblique cross-sectional views, respectively, of three additional alternatively configured flow chips, wherein the cross-sections are taken along the main channels;

[0036] Figure 4 is an image of the flow chip captured during particle preparation;

[0037] Figure 5 is a set of three images of the downstream portion of the flow chip showing the formation of liquid of the aqueous phase in the solvent phase;

[0038] Figure 6 is a set of three time-series images of the upstream portion of the flow chip, showing the formation of solvent phase droplets in the extraction phase in dripping mode;

[0039] Figure 7 is another set of three time-series images of the upstream portion of the flow chip, showing the formation of solvent phase droplets in the extraction phase in the spray mode;

[0040] Figure 8is a pair of images of the prepared particles, the left image shows the particles output from the flow chip, and the right image shows the particles after washing, where the signal corresponding to the labeled API (fluorescent dextran) is shown in red in the original color image. The results are described in Example 1;

[0041] Fig. 9 is an image captured in a comparative example using a flow chip in which the height in the main channel did not change during particle preparation;

[0042] Fig.10 yes Fig. 9 An image of particles resulting from the preparation shown, where the signal corresponding to the labeled API (fluorescent dextran) is shown in yellow in the original color image;

[0043] Fig.11 is an image of the flow chip captured during particle preparation, showing Figure 2a The effect of changes in the main channel height at the locations shown;

[0044] Fig.12 yes Fig.11 An image of the prepared particles shown, wherein the signal corresponding to the labeled API (fluorescent dextran) is shown in yellow in the original color image;

[0045] Fig.13 is a plan view of the flow chip showing the regions of the hydrophobic and hydrophilic surfaces of the channels;

[0046] Fig.14 The effect of varying the polymer molecular weight on the release characteristics of the particles produced by the present disclosure is shown. The results are described in Example 2;

[0047] Fig.15 The effect of varying the polymer shell thickness on the release characteristics of the particles produced by the present disclosure is shown. The results are described in Example 3;

[0048] Fig.16 The effect of varying the L:G ratio on the release characteristics of particles produced by the present disclosure, where the polymer used is PLGA, is shown. The effect is shown as a cumulative release plot over time. The results are described in Example 4;

[0049] Figure 17 shows the effect of varying the relative injection pressures of the aqueous and solvent phases in the disclosed process on the morphology of particles produced in the present disclosure. The results are described in Example 5;

[0050] Fig.18A comparison of the in vivo immune response after administration of egg white protein formulated in intact particles of the present disclosure is shown compared to when formulated in broken particles of the present disclosure or formulated with particles produced by the present disclosure (but without the pharmaceutical agent in the aqueous core). The results are described in Example 6;

[0051] Fig.19 A comparison of IgG responses in mice following immunization with R21 formulated in a soluble form (Ag-Sol) or encapsulated within particles according to the present disclosure (Ag-Par) is shown. The results are described in Example 7;

[0052] FIG20 shows (A) the morphology of heterogeneous particles produced by batch emulsification (vortexing all phases) and (B) a comparison of the in vitro release profiles of particles produced according to the present disclosure (right tracing; shown in red in the original color image) and heterogeneous particles (left tracing; shown in blue in the original color image). The results are described in Example 8.

[0053] Fig.21 Shown are the immune response kinetics of the vaccination regimens including encapsulated R21 compared to unencapsulated vaccines. For each of the tested regimens, antigen-specific IgG titers for the Cterm epitopes of the R21 vaccine were measured by ELISA in BALB / c mice (n=8 for each regimen). Titers were measured in tail blood collected weekly after immunization and expressed as median and interquartile range. The results are described in Example 9.

[0054] Fig. 22 It is shown that the peak antibody response of the encapsulated adjuvant-free R21 is not lower than that of the unencapsulated vaccine. The observed specific IgG peak for the Cterm epitope is plotted for each mouse (n=8 for each regimen). The fold change of the average peak log titer of the Cterm-specific response is estimated by the bootstrap method and recorded as the geometric mean fold change of the 95% CI (error bar) and the sampling distribution (density map). For each comparison, the non-inferiority interval when the change threshold is 0.5 times is shown as a faded rectangle.

[0055] Fig.23 The effect of adding adjuvant on the immune response kinetics of the encapsulated R21 regimen compared to the unencapsulated control is shown. Specific IgG against the Cterm epitope of the R21 vaccine in BALB / c mice (primary-Adj, n=16; other regimens n=8) was measured by ELISA. The titers were measured by collecting tail blood every week after immunization and expressed as median and interquartile range.

[0056] Fig.24The peak antibody response of encapsulated R21 and adjuvant is shown to be non-inferior to the unencapsulated control. The peak observed titer of specific IgG for the Cterm epitope is shown for each mouse (primary immunity-Adj, n=16; other schemes, n=8). The fold change of the average peak log titer of the Cterm-specific response was estimated by the bootstrap method and recorded as the geometric mean fold change of 95% CI (error bar) and sampling distribution (density map). For each comparison, the non-inferiority interval when the change threshold is 0.5 times is shown as a shaded rectangle.

[0057] Fig.25 Longitudinal Cterm-specific immunokinetic curves of different LCSS particle formulations are shown. Cterm-specific titers of R21 with adjuvant encapsulated in different LCSS particle formulations or unencapsulated were measured by ELISA (prime only, n=16; other regimens, n=8). Titers were measured weekly after vaccination, and the data for each mouse were connected with gray lines. Group-level responses from generalized additive model (GAM) fitting are shown as colored lines in the original color image, and 95% CI are represented by bands.

[0058] Fig.26 The estimated value of peak titer and the time to reach 50% peak titer are shown. The group level for Cterm-specific IgG reaction from the generalized additive model (GAM) fitting is shown as a colored line in the original color image, and 95% CI is represented by a band. The vertical line represents the time required to reach 50% peak titer. The geometric mean peak antibody titer of 10,000 simulated samples (fading points of each simulated sample) and the average time to reach 50% peak titer have 95% confidence intervals (shaded areas) and 95% marginal CI (error bars).

[0059] Fig. 27 The fold changes in peak antibody titers and additional immune response delays for different LCSS particle formulations compared to unencapsulated controls are shown. Geometric mean fold changes in Cterm-specific peak antibody titers and mean additional delays to reach 50% Cterm-specific antibody peak titers for different LCSS particle formulations compared to unencapsulated controls. Error bars represent 95% CIs and density plots represent simulated sampling distributions. Non-inferiority intervals with a 0.5-fold change threshold are shown as shaded rectangles.

[0060] Fig.28A summary of the design of the in vivo immunogenicity and malaria challenge experiments is shown, comparing the efficacy of the prime + particle regimen to the prime only regimen. BALB / c mice were immunized IM as shown. Injections were performed using a 100 μL Hamilton glass syringe sterilized with ethanol and a 25G needle. The injection volume was 50 μL, with 0.5% carboxymethylcellulose in PBS as a viscosity enhancer to prevent LCSS particles from aggregating in the syringe. Challenges were performed in two attack periods by injecting 1000 P. falciparum sporozoites into the tail vein of each mouse. The peak time window shown here corresponds to the 95% CI of the time when Cterm-specific immunogenicity reached 50% of the peak titer.

[0061] Fig.29 Shown are the immune response kinetics of different R21 / adjuvant regimens in short-term research attacks. For each of the three regimens tested, specific IgG for the Cterm epitope of the R21 vaccine in BALB / c mice (prime / boost regimen, n=8; other regimens, n=16) was measured by ELISA. Titers were measured in tail blood collected weekly after vaccination until the attack phase, expressed as median and interquartile range. The vertical dotted line represents the booster timing of the prime / boost regimen.

[0062] Fig.30 Peak antibody Cterm-specific responses in the prime and short PAR regimens are shown to be superior to unencapsulated prime and prime / boost regimens. Specific IgG peak titers observed for the Cterm epitope for each mouse (all regimens, n=8) are shown. The fold changes of the average peak log titers of Cterm-specific responses were estimated by bootstrapping and recorded as geometric mean fold changes of 95% CI (error bars) and sampling distributions (density plots).

[0063] Fig.31 The immune response kinetics of different R21 / adjuvant schemes in short-term research attacks are shown. For each of the three schemes tested, BALB / c mice (prime / boost scheme, n=8; other schemes, n=16) were measured by ELISA for specific IgG against the Cterm epitope of the R21 vaccine. Tail blood titers were collected weekly after vaccination until the attack phase, expressed as median and interquartile range. The vertical dotted line represents the booster timing of the prime / boost scheme.

[0064] Fig.32The peak antibody Cterm-specific response of the primary and mid-PAR regimens is shown to be superior to the unencapsulated primary regimen. The peak observed titers of specific IgG for the Cterm epitope observed for each mouse (prime / boost regimen, n=8; other regimens, n=16) are shown. The fold changes of the average peak log titers of Cterm-specific reactions were estimated by the bootstrap method and recorded as the geometric mean fold changes of 95% CI (error bars) and sampling distribution (density plots).

[0065] Fig.33 It is shown that compared with only the initial immunization, the initial immunization + short PAR is more protective and delays the time of malaria infection. BALB / c mice were immunized with different schemes (initial immunization / boosting, n=8; others, n=16) and challenged by intravenous injection of 1000 sporozoites. Attack the initial immunization / boosting mice 2 weeks after the booster immunization (4 weeks after the first immunization), while other immunization schemes attacked mice in two attack periods 3 or 4 weeks after immunization, with n=8 for each attack per scheme. A one-week blood stage parasitemia monitoring was carried out starting from the 5th day after the attack. The Kaplan-Meier curve of infection (1% parasitemia) for each scheme was drawn, and the data of the two attack periods were summarized. The table shows the risk ratio (estimated by Cox regression) of each scheme compared with the initial immunization scheme. The risk ratio is displayed with its respective 95%CI and p value.

[0066] Fig.34 It is shown that compared with only the primary immunization, the primary immunization + PAR is more effective in delaying the time of malaria infection. BALB / c mice were immunized with different schemes (primary immunization / boosting, n=8; others, n=16) and challenged by intravenous injection of 1000 sporozoites. Two weeks after the booster immunization (5 weeks after the first immunization), the mice receiving the primary immunization / boosting scheme were attacked, while the other schemes attacked the mice in two attack periods 5 or 6 weeks after immunization, with n=8 for each scheme each time. Blood stage parasitemia was monitored from the 5th day after the attack. The Kaplan-Meier curve of the attack period infection (1% parasitemia) collected for each scheme was drawn. The table shows the risk ratio (estimated by Cox regression) of each scheme compared with the primary immunization scheme. The risk ratio and its respective 95%CI and p value are displayed. DETAILED DESCRIPTION

[0067] Figure 1 A flow chip 1 for preparing particles is shown, wherein the particles include an aqueous core encapsulated by a polymer shell. The flow chip 1 is a material body in which a channel is formed. The channel is arranged from the upstream end to the downstream end as follows. The channel has a planar extent. The width of the channel is defined along the direction of the planar extent, and the height of the channel is defined along a direction perpendicular to the planar extent.

[0068] The flow chip 1 has a main channel 10 in which particles are formed. As described below, various liquids flow through the main channel 10. Figure 1 In the diagram, the upstream end is on the left and the downstream end is on the right.

[0069] The main channel 10 includes an aqueous phase inlet section 11 at the upstream end. In use, the aqueous phase flows into the flow chip 1 through the aqueous phase inlet section 11. As discussed further below, the aqueous phase includes a pharmaceutical agent and an aqueous solvent.

[0070] The flow chip 1 has a solvent phase inlet channel 30 on the opposite side of the main channel 1. In use, the solvent phase flows into the flow chip 1 through the solvent phase inlet channel 30. As discussed further below, the solvent phase comprises a polymer and a non-aqueous solvent.

[0071] The main channel 10 includes a solvent phase intersection section 12, to which the aqueous phase inlet section 11 and the solvent phase inlet channel 30 lead. The solvent phase inlet channel 30 extends at 90° to the aqueous phase inlet section 11, so that the solvent phase intersection section 12 provides a 90° intersection. In use, the aqueous phase and the solvent phase flowing into the solvent phase intersection section 12 form aqueous phase droplets in the solvent phase.

[0072] The main channel 10 includes a transfer section 13 extending from a solvent phase junction section 12 .

[0073] The transport section 13 includes a solvent nozzle section 14 adjacent to and downstream of the solvent phase intersection section 12. The width of the solvent nozzle section 14 increases with the distance from the solvent phase intersection section 12. In particular, the solvent nozzle section 14 includes a neck section 15 having a constant width and an expansion section 16 downstream of the neck section and having a width that increases with the distance from the solvent phase intersection section 12. In use, the solvent nozzle section 14 facilitates the formation of aqueous phase droplets in the solvent phase.

[0074] The transport section 13 includes a flow section 17 downstream of the solvent nozzle section 14 , the flow section 17 having a width smaller than the width of the solvent nozzle section 14 , thereby providing a constriction in the main channel 10 .

[0075] The flow chip 1 has an extraction phase inlet channel 40 disposed on the opposite side of the main channel 10. In use, the extraction phase flows into the flow chip 1 through the extraction phase inlet channel 40. As discussed further below, the extraction phase includes an extraction solvent for extracting the non-aqueous solvent from the solvent phase.

[0076] The main channel 10 includes an extract phase intersection section 18, into which the transport section 13 and an extract phase inlet channel 40 lead. The extract phase inlet channel 40 extends at 90° to the transport section 13, so that the extract phase intersection section 18 provides a 90° intersection. In use, the solvent phase and the extract phase flowing into the solvent phase intersection section 18 form solvent phase droplets encapsulating aqueous droplets in the extract phase.

[0077] The main channel 10 includes an extraction section 19 extending from the extraction phase junction section 18 .

[0078] The extraction section 19 includes an extraction nozzle section 20 adjacent to and downstream of the extraction phase junction section 18. The width of the extraction nozzle section 20 increases with the distance from the extraction phase junction section 18. In particular, the extraction nozzle section 20 includes a neck section 21 having a constant width and an expansion section 22 downstream of the neck section 21 and increasing in width with the distance from the extraction phase junction section 18. In use, the extraction nozzle section 20 facilitates the formation of solvent phase droplets in the extraction phase.

[0079] The extraction section 19 comprises a flow section 23 downstream of the extraction nozzle section 20 , the width of the flow section 23 being smaller than the width of the extraction nozzle section 20 , thereby providing a constriction in the main channel 10 .

[0080] In use, the non-aqueous solvent of the aqueous phase is extracted in the extraction stage 19 and subsequently particles are formed having an aqueous core formed by droplets of the aqueous phase and a shell formed by the polymer.

[0081] The main channel 10 comprises an output section 24 downstream of the extraction section 19, having a width greater than the width of the extraction section 19. In use, particles are collected in the output section 24. The output section 24 has an outlet 25 through which the extraction phase and particles can be extracted from the flow chip 1.

[0082] The main channel 10 has an increase in height which aids in the preparation of the particles, as will now be described.

[0083] Figure 2a and Figure 2b Two alternative configurations are shown in which the main channel 19 increases in height where the transfer section 13 leads to the extraction phase junction section 18. Figure 2a In an alternative arrangement, the increase in height occurs along a line projecting downstream, i.e. into the extraction phase junction section 18. In particular, the line is formed by two straight line segments (i.e. V-shaped) intersecting at one point, although alternatively the line may be curved. Figure 2b In the alternative structure, the increase in height occurs along a straight line.

[0084] Figure 3a to Figure 3cA second alternative is shown, whereby the main channel 19 increases in height downstream of the location where the transfer section 13 enters the extraction phase junction section 18 .

[0085] exist Figure 3a In an alternative structure, the increase in height occurs at the location where the neck section 21 of the extraction nozzle section 20 intersects the expanded section 22 of the extraction nozzle section 20.

[0086] exist Figure 3b In an alternative structure, the increase in height occurs where the expansion section 22 of the extraction nozzle section 20 intersects the flow section 23 of the transfer section 19.

[0087] exist Figure 3c In an alternative structure, the increase in height occurs at the intersection of the flow section 23 and the output section 24 of the transfer section 19.

[0088] exist Figure 3a to Figure 3c In each of the alternative configurations, the increase in height occurs along a line projecting downstream, i.e. into the extraction phase junction section 18. In each case, the line is formed by two straight line segments (i.e. V-shaped) meeting at a point, although alternatively the line may be curved. Figure 3a to Figure 3c In each of the alternative configurations, the increase in height may alternatively occur along a straight line.

[0089] exist Figure 2a , Figure 2b and Figure 3a to Figure 3c In the embodiment, the height variation of the main channel 10 is achieved by a step in one of the surfaces opposite in the height direction of the main channel. However, alternatively, the height variation of the main channel 10 can also be achieved by a step on each surface opposite in the height direction of the main channel.

[0090] Now refer to Figure 4 to describe a method for preparing particles using a flow chip, wherein Figure 4 It shows that Figure 2a Particle formation in flow chip 1 shown in cross section.

[0091] The aqueous phase flows through the aqueous phase inlet section 11 of the main channel 10 .

[0092] The solvent phase flows through the solvent phase inlet channel 30 and intersects with the aqueous phase at the solvent phase intersection section 12 of the main channel 10. Aqueous phase droplets are formed in the solvent phase, such as Figure 5 shown.

[0093] When the aqueous phase intersects with the solvent phase at the solvent phase intersection section 12, the two phases are immiscible (completely or partially) so that substantially no mixing between the two phases occurs. The solvent phase applies a shear strain to the aqueous phase, and the shear strain is balanced on each side of the aqueous phase because the solvent phase inlet channel 30 is located on the opposite side of the main channel 10. Once the interfacial tension is overcome, this provides the formation of droplets. The aqueous phase droplets can be formed in a dripping manner, forming an emulsion of aqueous droplets with high monodispersity inside the solvent phase. The droplets flow in an equidistant manner along the transmission section 13.

[0094] The solvent nozzle section 14 helps the aqueous phase to form droplets in the solvent phase, as described below. The solvent nozzle section 14 provides a flow focusing geometry after the solvent phase intersection section 12. Specifically, the increase in width in the solvent nozzle section 14, especially in the expansion section 16 of the solvent nozzle section 14, creates a velocity gradient, wherein the highest velocity is concentrated in the neck section 15 of the solvent nozzle section 14. This velocity gradient increases the shear strain applied to the aqueous phase in this solvent phase and causes the aqueous phase to break up into droplets in a reproducible manner. This allows the size distribution of the droplets to be tight.

[0095] The extraction phase flows through the extraction phase inlet channel 40 in the flow chip 1 .

[0096] The solvent phase entrained with the aqueous phase droplets flows through the transport section 13 of the main channel 10 and intersects with the extraction phase at the extraction phase intersection section 18 of the main channel 10. The solvent phase droplets encapsulating the aqueous phase droplets are formed in the extraction phase, such as Figure 6 shown.

[0097] When the solvent phase intersects with the extraction phase at the extraction phase intersection section 18, the two phases are immiscible (completely or partially miscible) so that mixing between the two phases does not occur substantially. The extraction phase applies a shear strain to the solvent phase, and the shear strain is balanced on each side of the solvent phase because the extraction phase inlet channel 40 is located on the opposite side of the main channel 10. Once the interfacial tension is overcome, this provides the formation of solvent phase droplets. The solvent phase droplets that encapsulate the aqueous phase droplets themselves can be formed in a dripping manner or a spraying manner, as further described below. In a general sense, the formation process relies on shear forces in a manner similar to the process of forming aqueous phase droplets in the solvent phase, but the solvent phase droplets formed encapsulate the aqueous phase droplets, thereby forming a double emulsion. The droplets flow along the extraction section 19 in an equidistant manner.

[0098] The extraction nozzle segment 20 facilitates the formation of droplets of the dissolved phase in the extraction phase in a manner similar to the action of the solvent nozzle segment 14, as described below. The extraction nozzle segment 20 provides a flow focusing geometry after the extraction phase junction segment 18. Specifically, the increase in width in the extraction nozzle segment 20, particularly the expansion segment 22 of the extraction nozzle segment 20, creates a velocity gradient, wherein the highest velocity is concentrated in the neck segment 21 of the extraction nozzle segment 20. This velocity gradient increases the shear strain exerted on the solvent phase in this extraction phase and causes the solvent phase to break up into droplets in a repeatable manner. This allows for the reliable formation of solvent phase droplets that encapsulate aqueous phase droplets.

[0099] The formation of solvent phase droplets depends on the flow rate of the solvent phase, as described below.

[0100] In one case, if Figure 6 As shown, after passing through the extraction nozzle section 20, solvent phase droplets may be formed in a dripping manner immediately.

[0101] Alternatively, in another case, such as Figure 7 As shown, the solvent phase may produce longer threads, which cause the liquid threads to break due to Rayleigh-Plateau instabilities, and finally form droplets through the jet. In this case, the droplets may form in the flow section 23 downstream of the extraction nozzle section 20, where the contraction accelerates the flow. To summarize the droplet formation mechanism, the disturbance of the solvent phase thread caused by the passage of the internal droplets of the aqueous phase produces a sinusoidal instability at the interface between the flow of the solvent phase and the external flow of the extraction phase. Due to the surface tension between the two phases, it causes the thread of the solvent phase to break into droplets surrounding the respective aqueous phase droplets.

[0102] To enable the particles to be used in drug delivery, a thick shell and high productivity are both desirable. These goals can be achieved by using high flow rates and solvent phases with relatively high viscosities, as well as having the highest concentration of dissolved polymer compatible with microfluidic processing. Figure 7 In the exemplary ejection mode, the extraction phase junction section 18 is advantageously used because it has a better throughput for viscous flow.

[0103] Under these conditions, the solvent phase and entrained aqueous droplets are confined in a jet passing through the extraction nozzle segment 20, decelerated in the extraction nozzle segment 20, and then accelerated again at the subsequent contraction of the flow segment 23. This, combined with the passage of the aqueous phase droplets within the solvent phase jet and the continuous contraction-expansion-contraction channel, causes instabilities in the solvent jet, causing the solvent jet to break into solvent phase droplets around each inner droplet of the aqueous phase. Since the formation of solvent phase droplets is triggered by the passing aqueous phase inner droplets, this improves the encapsulation efficiency and throughput of the production. This is better than letting the solvent phase drip and synchronizing the dripping with the passage of the aqueous phase inner droplets, although this is also possible.

[0104] The variation in height of the main channel 10 also has an effect, but this will be described below.

[0105] The solvent phase droplets flow through the extraction section 19 into the output section 24. Thereafter, the droplets can be collected from the output section 24 into another container.

[0106] The non-aqueous solvent of the aqueous phase is extracted from the solvent phase droplets, so that the particles formed have an aqueous core formed by the aqueous phase droplets and a shell formed by the polymer. The extraction can be performed by liquid phase extraction into the extraction phase and / or by evaporation.

[0107] Extraction begins in the extraction section 19. Depending on the length of the extraction section, extraction may occur entirely in the extraction section 19. Alternatively, extraction may continue while the droplets are in the output section 24, and / or subsequently after the droplets have been collected from the flow chip 1, for example in an open container such as a petri dish.

[0108] In one example, the droplets can be left in an open container in the extraction phase for extraction of the solvent by evaporation and then washed for liquid extraction, especially when the solvent used is miscible with water (e.g., in the case of DMC, which is 13% miscible with water). After a predetermined time selected to ensure that all droplets are converted into particles with a solid shell, in this example, 48 hours, the particles can be collected.

[0109] Any suitable collection method can be used. For example, the particles can be collected by centrifugation. The particles can be collected by filtration. The particles can be collected by evaporating the liquid medium in which the particles were initially prepared, such as by evaporating the extract phase. Those skilled in the art will appreciate that the particles can be prepared and isolated on a commercially relevant scale. In some embodiments, at least 10 1 10 2 10 3 10 4 10 5 10 6 10 7 108 or 10 9 The collected particles may be optionally washed and / or redispersed, for example using an aqueous carrier solution as defined herein, such as water.

[0110] The pellet can be used after multiple washes by centrifugation and replacement of the supernatant. Figure 8 Particles collected in this manner are shown, with the left side showing particles in an open container and the right side showing particles after centrifugal washing.

[0111] The particles may be formulated into a pharmaceutical composition comprising the particles and one or more pharmaceutically acceptable excipients, diluents or adjuvants.

[0112] The height variation of the main channel 10 improves droplet formation, as described below.

[0113] As a comparison, Fig. 9 A comparative example using a flow chip 1 in which the height of the main channel 10 is not changed is shown. In this case, there is compression of the solvent phase droplets in the extraction phase between the surfaces facing each other in the height direction of the main channel 10. As a result, Fig. 9 As shown, flow along the extraction section 19 is deformed and surfaces become wetted, potentially leading to subsequent blockage or disruption. Fig. 9 Also shown is the tailing of the core towards the tail of the solvent phase droplet, resulting in unloading of the particle.

[0114] Fig.10 An example of the particles prepared in this comparative example is shown in . It can be seen that the particles formed an unloaded and irregular double emulsion. There are many empty polymer droplets, so the content of the agent is reduced.

[0115] In contrast, Fig. 9 shows the use of the main channel 1μ in Figure 2a An example of a flow chip 1 is shown in which the height of the transport section 13 is not varied at the location where it leads to the extraction phase junction section 18. The effect of the variation in the height of the main channel 10 is to eliminate the compression that occurs in the comparative example. This prevents the aqueous phase droplets from being dragged into the tail of the solvent phase outer droplets. This also limits the wetting of the transport section, such as Fig.11 Thus, the double emulsion remains stable and can be collected without losing its core content agent.

[0116] Furthermore, the fact that double emulsions can be formed in the ejection mode also seems to be due to the fact that the continuous contraction and expansion of the droplet when it passes through this geometry will trigger the formation of the double emulsion.

[0117] Fig.12 Shows Fig.111. An example of particles prepared in the example of . It can be seen that the particles have good loading and monodispersity. Since the solvent phase includes an extractable solvent that can form a solid shell, the possibility of clogging when the extraction starts in the flow chip 1 is reduced. By preventing particles of the solvent phase from contacting the surface of the extraction section 19 and providing more space for solvent phase droplets to flow in the extraction phase, the risk of interfering with the production process is reduced.

[0118] It was also tested to locate the elevation change of the main channel 10 downstream of the location where the transfer section 13 leads to the extraction phase intersection section 18. Figure 3a to Figure 3c Flow chip 1 is shown, and the results show that it produces a similar effect of protecting newly formed double emulsions.

[0119] However, positioning the height change of the main channel 10 at the position where the transmission section 13 leads to the extraction phase intersection section 18 helps to slow down the flow of the solvent phase, which is conducive to the formation of solvent phase particles when the inertial force is too high (i.e., the flow is too high). Therefore, for some schemes, this is a more preferred design.

[0120] Figure 1 The dimensions of the flow chip 1 shown and non-limiting preferred ranges are as follows. In particular, these dimensions are such that the channels in the flow chip are not capillary, i.e. the flow chip 1 is not a capillary co-flow microfluidic device. In all ranges below, the upper and lower limits can apply independently.

[0121] · Aqueous phase inlet section 11:

[0122] Width: 20 μm (preferably in the range of 5 μm to 200 μm)

[0123] Height: 50 μm (preferably in the range of 5 μm to 250 μm)

[0124] Solvent phase inlet channel 30:

[0125] Width: 40 μm (preferably in the range of 5 μm to 500 μm)

[0126] Height: 50 μm (preferably in the range of 5 μm to 250 μm)

[0127] Neck section 15 of solvent nozzle section 14:

[0128] Width: 15 μm (preferably in the range of 5 μm to 200 μm)

[0129] Length: 30 μm (preferably in the range of 10 μm to 200 μm)

[0130] The expansion section 16 of the nozzle section 14:

[0131] Length: 170 μm (preferably in the range of 100 μm to 500 μm)

[0132] Width: 150 μm (preferably in the range of 100 μm to 500 μm)

[0133] Angle (relative to the axis of the main channel 10): 22° (preferably in the range of 10° to 90°)

[0134] ·Flow section 17 of the transmission section 13:

[0135] Width: 40 μm (preferably in the range of 5 μm to 500 μm)

[0136] Transmission segment 17:

[0137] Length: 1 mm (preferably in the range of 0.1 mm to 5 mm)

[0138] ·Height change downstream of the main channel 10:

[0139] Height: 50 μm (preferably in the range of 5 μm to 250 μm)

[0140] · Change in height upstream of the main channel 10:

[0141] Height: 100 μm (preferably in the range of 20 μm to 500 μm)

[0142] · Changes in the height of the main channel:

[0143] 50 μm (preferably in the range of 15 μm to 250 μm)

[0144] The line where the main channel height changes in the case of protrusion downstream:

[0145] Extending along the main channel 10: 25 μm (preferably in the range of 0 μm to 100 μm)

[0146] Extraction phase inlet channel 40:

[0147] Width: 80 μm (preferably in the range of 20 μm to 500 μm)

[0148] The height is the same as the main channel 10, so:

[0149] When the height change of the main channel 10 is located at the position where the transmission section 13 leads to the extraction phase intersection section 18, it is 100 μm (preferably in the range of 20 μm to 500 μm)

[0150] 50 μm (preferably in the range of 5 μm to 250 μm) when the height variation of the main channel 10 is located downstream of the position where the transfer section 13 leads to the extraction phase intersection section 18

[0151] Neck section 21 of extraction nozzle section 20:

[0152] Width: 30 μm (preferably in the range of 10 μm to 200 μm)

[0153] Length: 50 μm (preferably in the range of 10 μm to 200 μm)

[0154] · Enlarged section 22 of extraction nozzle section 20:

[0155] Length: 200 μm (preferably in the range of 100 μm to 1000 μm)

[0156] Width: 380 μm (preferably in the range of 100 μm to 1000 μm)

[0157] Angle (relative to the axis of the main channel 10): 41° (preferably in the range of 10° to 90°)

[0158] ·Flow section 23 of extraction section 19:

[0159] Width: 120 μm (preferably in the range of 10 μm to 200 μm)

[0160] Length: 250 μm (preferably in the range of 100 μm to 1000 μm)

[0161] Output section 24:

[0162] Length: 4 mm (preferably in the range of 1 mm to 10 mm)

[0163] Maximum width: 2 mm (preferably in the range of 1 mm to 1 μm)

[0164] Angle (relative to the axis of the main channel 1μ): 17° (preferably in the range of 5° to 90°)

[0165] The aqueous phase, solvent phase and extraction phase can be input into the flow chip 1 using a pressure-based system, wherein the pressure-based system enables the fluid to be injected from the corresponding reservoir at a constant pressure, or using a flow-based system (e.g., a syringe pump), wherein the fluid can be injected at a constant flow rate.

[0166] The flow rate can be measured by optical measurement from within the flow chip 1, by using a flow sensor (e.g., a thermal or mass flow sensor) placed between the reservoir of each phase and the flow chip 1 before the phases are input into the flow chip 1, or by calculation from the channel pressure and dimensions in the flow chip 1.

[0167] In order to maximize productivity, it is necessary to use the highest possible flow rate while maintaining the ability to generate droplets in the solvent phase intersection section 12 and the extraction phase intersection section 18. Ideally, to support the microfluidic emulsification process, the flow is laminar (using a relatively low Reynolds number Re, such as less than 2000), and dripping and jetting emulsification processes are possible (using relatively low capillary numbers (Ca) and Weber numbers (We)). This can be selected by controlling a combination of flow viscosity, interfacial tension and flow rate.

[0168] The flow rate is selected based on the core and shell sizes of the aqueous phase solution obtained after production as follows.

[0169] Increasing the ratio of the flow rate of the aqueous phase to the flow rate of the solvent phase increases the size of the aqueous phase droplets and thus increases the size of the resulting aqueous phase cores.

[0170] Increasing the ratio of the flow rate of the solvent phase to the flow rate of the extract phase increases the size of the solvent phase droplets, thereby increasing the size of the polymer shell.

[0171] Increasing the ratio of the flow rate of the aqueous phase to the flow rate of the extraction phase increases the probability of encapsulating two or more aqueous phase droplets within one solvent phase droplet, which is undesirable.

[0172] The flow rate is set above the minimum level of fluid flow through the flow chip 1 and the continuous phase at each intersection to apply sufficient shear stress to form an emulsion.

[0173] The flow rate is set above the maximum level that produces a continuous and stable phase jet without emulsification. The threshold estimate depends on the properties of the fluid, which vary depending on the particle formulation (different APIs, API buffers, polymers, polymer molecular weight, etc.), but can be derived experimentally for any chemical combination.

[0174] Figure 1 Some non-limiting but preferred flow rates for the flow chip shown are as follows:

[0175] Flow rate of aqueous phase: 0.25nL / s to 30μL / s

[0176] Solvent phase flow rate: 2.5nL / s to 150μL / s

[0177] Flow rate of extraction phase: 12nL / s to 3mL / s

[0178] The flow chip 1 can be manufactured using any suitable process.

[0179] A preferred option is to manufacture the flow chip 1 by multilayer moulding. This process can use moulds produced by multilayer soft lithography (eg available from Micrux microfluidix, Spain).

[0180] Multilayer soft lithography processes are known and are similar to single-layer processes. Briefly, for single-layer soft lithography, a layer of negative photoresist (SU-8) is spin-coated on a silicon wafer. It is then selectively exposed to UV using a mask comprising features having a first height. Another layer of negative photoresist is then spin-coated and exposed to UV through another mask comprising only a design having an extended height. The mold is developed to form a corresponding pattern as a positive mold. The positive mold is treated with trimethylchlorosilane to prevent adhesion of the material of the flow chip 1 (e.g., PDMS). The uncured material is then poured onto the mask and cured to carve out a channel (negative pattern) on a solid block of the flow chip 1 material. Inlets and outlets are punched out in the flow chip 1, and the design is sealed by bonding the block with the channel carved out to the underlying material of the flow chip 1, for example, using an O2 plasma cleaner.

[0181] The flow chip can be made of any suitable material. Typically, the flow chip includes a body that includes a moldable material or is composed of a moldable material. Exemplary materials include: polymers, such as PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), polycarbonate (PC) or cycloolefin copolymer (COC); and glass. When the flow chip includes a body that includes or is composed of glass, the glass can be treated, for example, to control the chemical properties of the solvent-accessible surface. For example, the glass surface can be treated with silane to control the hydrophobicity of the surface. Surfaces, such as silane modification of glass are well known in the art, and silane reagents are commercially available, such as from Gelest. Hydrophobic silanes include methyl silanes, linear or branched alkyl silanes, aromatic silanes, dialkyl silanes, etc. Fluorinated silanes can be used to further increase hydrophobicity. Hydrophilicity can be increased by modifying the glass surface with a silane reagent containing one or more functionalized hydrocarbon groups functionalized with hydrophilic functional groups. For example, alkanes functionalized with hydroxyl or amino groups can be used. Polymer surfaces can also be similarly modified. For example, PDMS itself is hydrophobic, but can be treated with hydrophilic polymers, such as polyvinyl alcohol, to increase its hydrophilicity. For example, after the chip is produced, a 1% solution of polyvinyl alcohol (PVA) in water can be introduced into the channels of the chip to increase the hydrophilicity of selected areas (other areas can be protected by injecting air); see, for example, Trantidou, T., Elani, Y., Parsons, E. et a1., Hydrophilic surface modification of PDMS for droplet microfluidics using a simple, quick, and robust method via PVA deposition, Microsyst Nanoeng 3, 16091 (2017).

[0182] As will be appreciated by those skilled in the art, the main body material is typically selected to be non-reactive with the solvents used in the disclosed methods (e.g., swell or dissolve in the presence of a solvent), particularly when in contact with a non-aqueous solvent. Thus, the material constituting the main body of the flow chip will typically not dissolve and / or swell when in contact with an organic solvent as described herein. For example, PDMS is compatible with solvents such as dimethyl carbonate.

[0183] In some embodiments, different parts of the chip may advantageously have different hydrophilicities. Fig.13As shown, the surface of the main channel 10 of the flow chip 1 upstream of the extraction phase intersection section 18 is generally hydrophobic (e.g., made of a hydrophobic material or treated to be hydrophobic). This may be beneficial because it prevents droplets of the pharmaceutical agent in the aqueous solvent generated as described herein from forming a post-wetting channel wall at the solvent phase intersection section 12.

[0184] Typically, the extraction solvent is hydrophilic, the surface of the extraction phase inlet channel 40 is hydrophilic, and the surface of the main channel 10 of the flow chip 1 at and downstream of the extraction phase junction section 18 is hydrophilic. This may be beneficial because it prevents the outer droplets of the non-aqueous solvent (which encapsulate the inner droplets of the aqueous solvent and the agent) from forming a post-wetting channel wall at the extraction phase junction section 18.

[0185] The flow chip can be produced by any suitable method for forming a three-dimensional structure from a solid material. When the body of the flow chip is made of a polymer, molding (e.g., soft lithography molding) is a suitable method. Alternatively, the flow chip can be manufactured by hot embossing (e.g., by hot embossing a polymer sheet). When the body of the flow chip is made of glass, etching is a suitable method. Different parts of the flow cell can be treated with different reagents, by using gas (e.g., air) bubbles to separate the areas in the flow cell that should not be treated from the fluid path of the cell body.

[0186] Aqueous phase

[0187] As described above, according to the methods of the present disclosure, the aqueous phase entrained in the solvent phase includes a pharmaceutical agent and an aqueous solvent.

[0188] The aqueous solvent of the aqueous phase may include deionized water and / or ultrapure water (eg, having a resistivity of at least 10 MΩcm, such as at least 15 MΩcm, such as at least 18 MΩΩcm) before adding any additional components (eg, buffer salts).

[0189] The aqueous solvent of the aqueous phase typically has a pH of about 4 to about 10. More typically, the aqueous solvent of the aqueous phase has a pH of about 5 to about 9, such as about 6 to about 8.

[0190] The aqueous solvent of the aqueous phase may optionally include one or more buffer salts. When present, the buffer salt is contained in the aqueous solvent of the aqueous phase. For some applications, no buffer is required, and any buffer may be omitted from the aqueous phase.

[0191] When used, preferred buffer salts include: Tris; phosphate; citric acid / Na2HPO4; citric acid / sodium citrate; sodium acetate / acetic acid; Na2HPO4 / NaH2PO4; glyoxaline / HCl; sodium carbonate / sodium bicarbonate; ammonium carbonate / ammonium bicarbonate; MES; Bis-Tris; ADA; aces; PIPES; MOPSO; Bis-Tris propane; BES; MOPS; TES; HEPES; DIPSO; MOBS; TAPSO; Trizma; HEPPSO; POPSO; TEA; EPPS; Trimethylglycine (Tricine); Glv-Gly; Bicine (Bicine); HEPBS; TAPS; AMPD; TABS; AMPSO; CHES; CAPSO; AMP; CAPS and CABS. Additional salts include salts of anthoates, such as Mg(OH)2 and MgCO3, which are generally immiscible and suspended in the aqueous phase. Preferably, the buffer is pharmaceutically acceptable. Pharmaceutically acceptable buffers include phosphate / phosphoric acid, citrate / citric acid, acetate / acetic acid, histidine, lactate / lactic acid, tromethamine, gluconic acid / gluconate, aspartic acid / aspartate, tartaric acid / tartrate, succinic acid / succinate, malic acid / malate, fumaric acid / fumarate, and α-ketoglutaric acid / α-ketoglutarate. The most common buffer salts are selected from phosphate, citrate and acetate.

[0192] The selection of an appropriate buffer for the desired pH is routine for the skilled person and guidance is available at, for example, http: / / www.sigmaaldrich.com / life-science / core-bioreagents / biological-buffers / learning-center / buffer-reference-center.html. Buffer salts are preferably used in solution at a concentration of 1 mM to 1 M, preferably 1 mM to 100 mM, such as about 10 mM to about 50 mM.

[0193] The preferred aqueous solvent for the aqueous phase of the present disclosure is phosphate buffered saline (PBS). In some embodiments, the PBS includes NaCl and / or KCl, buffered to about 7.4 using a phosphate buffer. For example, the PBS may include 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4.

[0194] In some embodiments, the aqueous phase or the aqueous solvent of the aqueous phase includes one or more gelling agents. This can be beneficial for producing particles in which the aqueous core of the particles is a semisolid (e.g., in the form of a hydrogel). When such a gelling agent is not present or is only present in low concentrations, the aqueous core can be a liquid. Typically, the composition of the aqueous phase is controlled so that the aqueous core is a liquid.

[0195] When the aqueous phase includes a gelling agent, any suitable gelling agent may be used. Typically, the gelling agent is pharmaceutically acceptable. Gelling agents for use in accordance with the present disclosure may include tragacanth, pectin, xanthan gum, gellan gum, guar gum, gelatin, starch, carbomer, alginates, carrageenan, chitosan, poloxamer, poly(ethylene) oxide, linear or branched poly(ethylene) glycol (PEG) (e.g., chemically modified poly(ethylene) glycol modified with, for example, acrylate, norbornene, vinyl sulfone, maleimide and / or thiol functional groups), polycarbophil, gelatin, cellulose (e.g., hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hypromellose, hydroxyethyl cellulose (HEC), methyl cellulose, etc.), povidone, polyvinyl alcohol clay, polymeric gelling agents such as carbomer (e.g., carbomer 934P, carbomer 940, carbomer 941, etc.), bentonite, and the like.

[0196] Typically, when present, the gelling agent is used in a concentration of about 0.1% w / v to about 20% w / v, such as about 0.5% to about 10% w / v, such as about 1% to about 5% w / v in the aqueous phase. Such concentrations are suitable for preparing particles in which the aqueous phase core is a semi-solid.

[0197] The aqueous phase may include one or more stabilizers, such as trehalose and L-lysine.

[0198] The aqueous phase may include the agent in solution or suspension. Most commonly, the aqueous phase includes the agent in solution.

[0199] Any suitable pharmaceutical agent may be used. Pharmaceutical agents that may be incorporated into the aqueous phase are described in greater detail herein.

[0200] Typically, the agent is a hydrophilic agent. Typically, the agent is water soluble. Typically, the agent is soluble in a buffered saline solution having a pH of about 4 to about 10.

[0201] Solvent Phase

[0202] As described above, according to the method of the present disclosure, the solvent phase entrains the aqueous phase and includes a polymer and a non-aqueous solvent. The non-aqueous solvent is capable of dissolving the biodegradable polymer used for the polymer shell of the particles of the present disclosure. Therefore, the solvent phase produces the polymer shell of the particles produced according to the method of the present disclosure.

[0203] Typically, the non-aqueous solvent is an organic solvent.

[0204] Typically, the non-aqueous solvent is biodegradable. An example of a biodegradable solvent is dimethyl carbonate (DMC), which is hydrolyzed by water in vitro and metabolized to methanol and carbon dioxide in vivo. DMC is considered a green reagent.

[0205] Typically, the non-aqueous solvent is capable of dissolving polymers such as aliphatic polyesters, aromatic copolyesters, polyurethanes, polycarbonates, polyamides, poly(acid-amides), polyanhydrides, polysaccharides, and blends thereof or copolymers thereof. More typically, the non-aqueous solvent is a solvent for aliphatic polyesters and polyanhydrides.

[0206] Typically, the non-aqueous solvent is a solvent for polymers such as poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(butylene succinate), poly(p-dioxanone) (PPDO), poly(hydroxybutyrate) (PHB), poly(butylene adipate-co-butylene terephthalate) (PBAT), chitosan, cellulose, hyaluronic acid, and blends and copolymers thereof. More typically, the non-aqueous solvent is capable of dissolving poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polycaprolactone (PCL), polyanhydrides, and blends and copolymers thereof. More typically, the non-aqueous solvent is capable of dissolving poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA) and polycaprolactone (PCL), and blends and copolymers thereof. Most typically, the non-aqueous solvent is capable of dissolving poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), and polycaprolactone (PCL). For example, the non-aqueous solvent can dissolve (i.e., act as a solvent) a PLGA polymer comprising a blend of lactic acid and glycolic acid (L:G ratio) of 0:100 to 100:0, preferably 10:90 to 90:10, such as 25:75 to 75:25, for example 40:60 to 60:40.

[0207] Typically, the non-aqueous solvent is not a solvent for the material used to make the device described herein. This prevents the solvent from affecting the structural integrity of the device during operation of the disclosed method. For example, non-aqueous solvents typically cannot dissolve materials such as PDMS, glass, PMMA, polycarbonate, cyclic olefin copolymers, etc. Most typically, non-aqueous solvents do not dissolve PDMS.

[0208] Typically, non-aqueous solvents are immiscible or at most partially miscible with water, and thus are generally immiscible or at most partially miscible with the aqueous phase and / or extraction phase described herein. Typically, the solubility of the non-aqueous solvent in water is at most 50 g / 100 mL, for example at most 30 g / 100 mL, such as at most 20 g / 100 mL, for example at most 15 g / 100 mL, more preferably at most 10 g / 100 mL or less. For example, the solubility of DMC is 13.9 g / 100 mL.

[0209] Typically, the melting point of the non-aqueous solvent is lower than about 30°C, for example, lower than about 25°C, such as lower than 20°C, for example, less than 15°C, more preferably less than 10°C. This ensures that the solvent is not solid at conventional operating temperatures. Typically, the boiling point of the non-aqueous solvent is at least about 50°C, such as at least 60°C, for example at least 70°C, for example at least 80°C, for example at least 90°C or higher. This ensures that the solvent is liquid at conventional operating temperatures. For example, the melting point of DMC is about 2°C to 4°C, and the boiling point is about 90°C.

[0210] Typically, the non-aqueous solvent can be extracted into the aqueous solvent. By extracting the non-aqueous solvent into the aqueous solvent, the polymer dissolved therein solidifies, thereby forming a solid homogeneous shell around the aqueous core of the particle. Alternatively or additionally, the non-aqueous solvent can be removed by evaporation.

[0211] Exemplary solvents used in the present disclosure include dimethyl carbonate (DMC), methylene chloride (DCM), toluene and / or chloroform. Other solvents include n-hexane, ether, benzene, n-butyl alcohol, butyl acetate, carbon tetrachloride, cyclohexane, 1,2-ethylene dichloride, ethyl acetate, heptane, methyl tert-butyl ether, methyl ethyl ketone, pentane and ethylene dichloride. More typically, the solvent used is dimethyl carbonate (DMC).

[0212] It is within the ability of one skilled in the art to select an appropriate solvent for the desired polymer included in the particle shell produced herein: this is an operating parameter that can be determined by one skilled in the art.

[0213] Typically, the concentration of the polymer in the non-aqueous solvent is high. Typically, the concentration is as high as possible while being able to be processed by the device and the method disclosed herein. The use of a high concentration of polymer can be used to produce particles with a thick shell, which in turn can improve the stability of the particles produced in the disclosed method, such as in storage and / or once administered to a subject (e.g., for delayed release of an aqueous core). Any suitable concentration can be used in the present disclosure. For example, the concentration range of the polymer in the non-aqueous solvent can be about 0.5% to about 25% w / v (weight to volume), such as about 5% to about 20% w / v, such as about 8% to about 18% w / v, such as about 10% to about 15% w / v, for example, about 12.5% ​​w / v. Typically, higher concentrations can be used for polymers of lower molecular weight, and lower concentrations can be used for polymers of higher molecular weight. For example, a polymer (e.g., PLGA) with a molecular weight of about 5kDa to about 20kDa (e.g., about 7kDa to about 17kDa) can be used at a concentration of about 15% to about 20% w / v, such as about 17.5% w / v. A polymer (e.g., PLGA) having a molecular weight of about 20 kDa to about 40 kDa (e.g., about 24 kDa to about 38 kDa) can be used at a concentration of about 10% to about 15% w / v, for example, about 13% w / v. A polymer (e.g., PLGA) having a molecular weight of about 40 kDa to about 55 kDa (e.g., about 38 kDa to about 54 kDa) can be used at a concentration of about 8% to about 12% w / v, for example, about 10.5% w / v. A polymer (e.g., PLGA) having a molecular weight of about 55 kDa to about 70 kDa (e.g., about 54 kDa to about 69 kDa) can be used at a concentration of about 5% to about 10% w / v, for example, about 8.5% w / v.

[0214] Typically, the solvent phase includes one or more biodegradable polymers. Any suitable biodegradable polymer may be used in accordance with the present invention. The use of a biodegradable polymer is very advantageous because it prevents the accumulation of non-biodegradable polymers in the body when the particles produced according to the disclosed methods are administered to a subject, which accumulation may be toxic or harmful.

[0215] Typically, once administered to a subject as described herein, one or more biodegradable polymers are capable of biodegrading within a time frame of between about 1 day to about 1 year, such as about 1 week to about 6 months, such as about 2 weeks to about 4 months (e.g., about 2 weeks to about 10, 11, 12, 13 or 14 weeks), such as about 2 weeks to about 2 months. It is straightforward for one skilled in the art to determine the time frame for polymer biodegradability.

[0216] Typically, the biodegradable polymers suitable for use in the present disclosure have a melting temperature (T ) of about 20° C. to about 200° C., such as about 50° C. to about 150° C.m ). This can facilitate maintaining a sterile form of the composition comprising the polymer.

[0217] Typically, biodegradable polymers suitable for use in the present disclosure are soluble in organic solvents, such as dimethyl carbonate (DMC), dichloromethane (DCM), toluene and / or chloroform. Typically, biodegradable polymers suitable for use in the present disclosure are soluble in dimethyl carbonate (DMC). This can facilitate easy implementation or manufacture of the methods and devices disclosed herein.

[0218] Typically, the methods and resulting particles provided herein include only one type of polymer, or include a mixture of at most two or three polymers. More typically, only one type of polymer is used. The use of a single type of polymer can facilitate easy implementation or manufacture of the methods and devices disclosed herein. The use of a mixture or blend of polymers or copolymers enables the adjustability of the release characteristics of the particles produced according to the disclosed methods to be improved.

[0219] Typically, biodegradable polymers suitable for use in the present disclosure are selected from aliphatic polyesters, aromatic copolyesters, polyurethanes, polycarbonates, polyamides, poly(ester-amides), polyanhydrides, polysaccharides, and blends thereof or copolymers thereof.

[0220] More typically, biodegradable polymers suitable for use in the present disclosure are selected from aliphatic polyesters and polyanhydrides.

[0221] Typically, the biodegradable polymer suitable for use in the present disclosure is selected from poly(lactic acid-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(butylene succinate), poly(p-dioxanone) (PPDO), poly(hydroxybutyrate) (PHB), poly(butylene adipate-co-butylene terephthalate) (PBAT), chitosan, cellulose, hyaluronic acid, and blends thereof or copolymers thereof.

[0222] More typically, biodegradable polymers suitable for use in the present disclosure are selected from poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polycaprolactone (PCL) and polyanhydrides, and blends thereof or copolymers thereof. More typically, biodegradable polymers suitable for use in the present disclosure are selected from poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA) and polycaprolactone (PCL), and blends thereof or copolymers thereof. Most typically, biodegradable polymers suitable for use according to the present disclosure are selected from poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA) and polycaprolactone (PCL).

[0223] More preferably, the biodegradable polyester is poly(lactic-co-glycolic acid) (PLGA), or a blend thereof or a copolymer thereof. Preferably, the PLGA polymer comprises a blend of lactic acid and glycolic acid (L:G ratio) of 0:100 to 100:0, preferably 10:90 to 90:1v, such as 25:75 to 75:25, for example 40:60 to 60:40. Without being bound by theory, it is believed that when a PLGA polymer with a higher L:G ratio is used, the release of the liquid core from the solid shell of the particles of the present disclosure can be delayed. Therefore, in some embodiments, the L:G ratio is 50:50 to 100:0, for example 60:40 to 90:10, for example about 70:30 to about 80:20, for example about 75:25. Therefore, a person skilled in the art can control the release characteristics of the particles of the present disclosure by controlling the L:G ratio of the PLGA polymer used to form the polymer shell of the particles. This is shown in Examples 4 and Fig.16 Described in .

[0224] Typically, biodegradable polymers suitable for use in the present disclosure have an average molecular weight (e.g., weight average molecular weight) of about 1 kDa to about 250 kDa, e.g., about 5 kDa to about 100 kDa, e.g., about 10 kDa to about 50 kDa. The inventors have found that the release characteristics of the particles produced according to the disclosed method can be adjusted by controlling the average molecular weight of the polymer used to produce the polymer shell. Typically, the use of a biodegradable polymer with a higher average molecular weight can cause a longer delay before the release of the medicament from the liquid core of the polymer. The delay in this case can be an in vivo delay after the composition comprising the particles is applied to a subject, or can be measured or measured in vitro.

[0225] For example, using a PLGA with an average molecular weight of about 5 to 20 kDa can produce particles that release the core contents after about 15 to 30 days when measured in vitro. Using a PLGA with an average molecular weight of about 20 to 40 kDa can produce particles that release the core contents after about 16 to 35 days when measured in vitro. Using a PLGA with an average molecular weight of about 40 to 55 kDa can produce particles that release the core contents after about 20 to 40 days when measured in vitro. Using a PLGA with an average molecular weight of about 55 to 70 kDa can produce particles that release the core contents after about 25 to 45 days when measured in vitro.

[0226] As a particularly preferred example, the solvent phase may therefore include DMC as a non-aqueous solvent and PLGA with an average molecular weight of about 5 to 20 kDa as a polymer, preferably at a concentration of about 5% to about 20% w / v, such as about 12.5% ​​to about 17.5% w / v.

[0227] Extraction phase

[0228] As described herein, the disclosed method includes: forming droplets of an aqueous phase encapsulated by a solvent phase in an extraction phase; and extracting a non-aqueous solvent of the solvent phase, thereby producing particles.

[0229] The extraction phase comprises an extraction solvent. Any suitable extraction solvent may be used. A suitable extraction solvent is a solvent that is capable of extracting the non-aqueous solvent from the solvent phase of the droplet emulsion as described above.

[0230] Typically, the extraction solvent is non-organic. Typically, the extraction solvent is aqueous, although other polar solvents, such as ionic liquids, may also be used.

[0231] Typically, the extraction solvent is physiologically acceptable. This is beneficial because it means that trace amounts of the solvent can remain in the final particle product (e.g., as a medium holding the particles) and can be administered to a subject without harm.

[0232] The extraction solvent may include deionized water and / or ultrapure water (eg, having a resistivity of at least 10 MΩcm, such as at least 15 MΩcm, for example at least 18 MΩcm), before adding any other components (eg, buffer salts).

[0233] Typically, the extraction solvent has a pH of about 4 to about 10. More typically, the extraction solvent has a pH of about 5 to about 9, such as about 6 to about 8.

[0234] The extraction solvent may optionally include one or more buffer salts. For some applications, no buffer is required, and the extraction solvent may omit any buffer. When used, preferred buffer salts include: Tris; phosphate; citric acid / Na2HPO4; citric acid / sodium citrate; sodium acetate / acetic acid; Na2HPO4 / NaH2PO4; glyoxaline / HCl; sodium carbonate / sodium bicarbonate; ammonium carbonate / ammonium bicarbonate; MES; Bis-Tris; ADA; aces; PIPES; MOPSO; Bis-Tris propane; BES; MOPS; TES; HEPES; DIPSO; MOBS; TAPSO; Trizma; HEPPSO; POPSO; TEA; EPPS; trimethylglycine (Tricine); Gly-Gly; Bicine (Bicine); HEPBS; TAPS; AMPD; TABS; AMPSO; CHES; CAPSO; AMP; CAPS and CABS. Preferably, the buffer is pharmaceutically acceptable. Pharmaceutically acceptable buffers include phosphate / phosphoric acid, citrate / citric acid, acetate / acetic acid, histidine, lactate / lactic acid, tromethamine, gluconic acid / gluconate, aspartic acid / aspartate, tartaric acid / tartrate, succinic acid / succinate, malic acid / malate, fumaric acid / fumarate, and α-ketoglutaric acid / α-ketoglutarate. The most common buffer salts are selected from phosphate, citrate and acetate.

[0235] The selection of an appropriate buffer for the desired pH is routine for the skilled person and guidance is available, for example, at http: / / www.sigmaaldrich.com / life-science / core-bioreagents / biological-buffers / learning-center / buffer-reference-center.html. Buffer salts are preferably used in solution at a concentration of 1 mM to 1 M, preferably 1 mM to 100 mM, for example about 10 mM to about 50 mM.

[0236] The preferred extraction solvent is phosphate buffered saline (PBS). In some embodiments, the PBS includes NaCl and / or KCl, and is buffered to about 7.4 using a phosphate buffer. For example, the PBS may include 137 mM NaCl, 2.7 mm KCl, 8 mM Na2HPO4, and 2 mM KH2PO4.

[0237] The extraction solvent may contain a surfactant. The surfactant may be used to help prevent the droplets from agglomerating without interfering with the emulsification process.

[0238] Any suitable surfactant can be used. Typically, the surfactant is water soluble and physiologically acceptable. Using a physiologically acceptable surfactant is advantageous because, in some embodiments, it can avoid the need to remove all trace amounts of surfactant from the particles prior to administration to a subject.

[0239] Typically, the surfactant is selected from anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants. Anionic surfactants are typically organic salts that dissociate at high pH to form long-chain anions with surface activity. Anionic surfactants typically contain carboxylates, sulfonates, phosphates or sulfates. Examples include alkyl sulfates, such as ammonium lauryl sulfate, sodium lauryl sulfate (sodium dodecyl sulfate, SLS or SDS), and related alkyl ether sulfates, sodium lauryl ether sulfate (sodium dodecyl ether sulfate or SLES) and sodium myristyl alcohol ether sulfate. Other anionic surfactants include docusate (sodium dioctyl sulfosuccinate), perfluorooctane sulfonate / salt (PFOS), perfluorobutane sulfonate / salt, alkyl aryl ether phosphate / salt, alkyl ether phosphate / salt and carboxylate / salt, such as sodium stearate, sodium lauroyl creatine, perfluorononanoate and perfluorooctanoate (PFOA or PFO).

[0240] Cationic surfactants are generally positively charged substances that often also have antimicrobial properties. Examples include phosphatidylcholine; octenidine dihydrochloride; permanently charged quaternary ammonium salts: cetrimonium bromide (CTAB), cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzylcarboxylate chloride (BZT), dimethyldioctadecyl ammonium chloride, and dioctadecyldimethylammonium bromide (DODAB).

[0241] Amphoteric surfactants exhibit dissociation of anions and cations and include betaines, (e.g., sulfobetaines) and natural substances such as amino acids and phospholipids. Other examples include sulfonates, such as in betaine CHAPS (3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate) and cocamidopropyl hydroxysulfobetaine, betaines such as cocamidopropyl betaine with ammonium carboxylates, and phospholipids such as phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin. Lauryl dimethylamine oxide and myristic amine oxide are two other common examples.

[0242] Nonionic surfactants include esters such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate, ), polyoxyethylene 15 hydroxystearate (e.g. Macrogol 15 hydroxystearate, Solutol ), polyoxyethylene castor oil derivatives (e.g. EL, ELP, RH40), polyoxyethylene stearate (e.g. ), sorbitan fatty acid esters (e.g. ), polyoxyethylene alkyl ethers (e.g. ) and polyoxyethylene nonylphenol ether (such as ).

[0243] Additional examples include ethoxylates (e.g., fatty alcohol ethoxylates, narrow range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether); alkylphenol ethoxylates (APE or APEO) (e.g., nonoxynol ether and Triton X-100); fatty acid ethoxylates; ethoxylated amines and / or fatty acid amides (e.g., polyethoxylated tallow amine; cocoyl monoethanolamine; cocoyl diethanolamine); end-blocked ethoxylates, such as poloxamers; fatty acid esters of polyols; fatty acid esters of glycerol (e.g., glyceryl monostearate, glyceryl monolaurate); fatty acid esters of sorbitol (e.g., sorbitol monolaurate, sorbitol monostearate, and sorbitol tristearate; Tweens, such as Tween 20, Tween 40, Tween 60, Tween 70, Tween 80, Tween 90, Tween 100, Tween 110, Tween 120, Tween 130, Tween 140, Tween 150, Tween 160, Tween 170, Tween 180, Tween 190, Tween 210, Tween 230, Tween 240, Tween 250, Tween 260, Tween 270, Tween 280, Tween 290, Tween 300, Tween 310, Tween 320, Tween 330, Tween 340, Tween 350, Tween 360, Tween 370, Tween 380, Tween 3 80); fatty acid esters of sucrose and alkyl polyglycosides (e.g., decyl glucoside, lauryl glucoside, octyl glucoside).

[0244] A particularly suitable surfactant for the present disclosure is polyvinyl alcohol (PVA). Typically, PVA can have a molecular weight of about 2000 to about 20000 g / mol, such as about 5000 to about 15000 g / mol, for example about 8000 to about 12000 g / mol, for example about 9000 to about 10000 g / mol. Typically, PVA can be about 50% to about 99% hydrolyzed, for example about 60% to about 90% hydrolyzed, for example about 70% to about 85% hydrolyzed, such as about 80% hydrolyzed.

[0245] The concentration of the surfactant in the extraction solvent may be about 0.01% to about 20% w / v, for example about 0.1% to about 10% w / v, for example about 1% to about 5% w / v, such as about 3% w / v.

[0246] In an exemplary embodiment, the surfactant may be PVA having a molecular weight of about 9000 to 10000 g / mol, about 80% hydrolyzed, and a concentration of about 3% w / v in the extraction solvent. The extraction solvent may be, for example, PBS as defined herein.

[0247] Additional components may be added to the extraction solution. For example, thickeners such as glycerol, poly(ethylene) glycol, carboxymethyl cellulose, xanthan gum may be incorporated. In the aqueous context, any gelling agent disclosed may generally be included in the extraction solvent, typically in a low concentration below its gelling concentration.

[0248] It is also possible to further include an osmotic pressure regulator, such as a salt. Examples include NaCl, KCl, MgCl2, phosphates, etc. In general, any buffer salt disclosed herein can be used to adjust the osmotic pressure of the extraction solvent. This can be used to match the osmotic pressure of the aqueous core of the particle.

[0249] Particles

[0250] As can be appreciated from the above discussion, particles produced according to the present disclosure can be contrasted with particles produced by conventional double emulsion methods, such as an aqueous solution of a pharmaceutical agent ultrasonically treated in an organic polymer solution to form a water-in-oil (W / O) emulsion, which is then further combined and homogenized in another aqueous solution to form a water-in-oil (W / O / W) double emulsion. As described in the examples, particles formed by this method are generally not monodisperse, spherical, and / or stable, and generally lack a well-defined aqueous core encapsulated by a solid polymer shell, which is a characteristic of the particles of the present disclosure.

[0251] It is obvious that the aqueous core is derived from the aqueous phase discussed herein. Therefore, when using the apparatus and / or method disclosed herein to produce these particles, the composition of the aqueous core of the particles provided herein can be controlled by controlling the composition of the aqueous phase used in the apparatus and method. In some embodiments, the composition of the aqueous core is the same as the composition of the aqueous phase. In some embodiments, the composition of the aqueous phase is the same as the composition of the aqueous core of the particles.

[0252] Thus, the aqueous core may be a liquid, or a semisolid in the form of a hydrogel. When the aqueous core is a gel, the aqueous core typically includes one or more gelling agents as described in more detail herein in the context of the aqueous phase. Typically, the aqueous core is a liquid.

[0253] The aqueous core may include the agent in solution or suspension. Most commonly, the aqueous core includes the agent in solution. The agents are described in more detail herein.

[0254] The aqueous core typically has a pH of about 4 to about 10, and may include one or more buffer salts, as discussed in greater detail herein, particularly in the context of the aqueous phase.

[0255] As is evident from the above discussion, the methods provided herein produce uniform particles in which the aqueous core is well defined.

[0256] Typically, the average diameter of the aqueous core of the particles of the present disclosure is from about 1 μm to about 300 μm. More typically, the average diameter of the core of the particles of the present disclosure is from about 3 μm to about 150 μm, for example from about 10 μm to about 100 μm, such as from about 30 μm to about 70 μm, for example from about 40 μm to about 55 μm. There are many methods that can be used to measure the diameter of the core of the liquid / shell particles, such as SEM and optical (e.g., fluorescence) microscopy.

[0257] Typically, the aqueous core is highly spherical. This is defined in more detail herein. Preferably, the minimum diameter of the aqueous core is at least 70% of the aqueous core maximum diameter. More typically, the minimum diameter of the aqueous core can be at least 90% of the aqueous core maximum diameter, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, at least 99.99% or at least 99.999% of the aqueous core maximum diameter. Preferably, when, for example, measured by a scanning electron microscope (SEM) or an optical microscope (such as a fluorescence microscope), the minimum diameter of the aqueous core is indistinguishable from the maximum diameter of the aqueous core.

[0258] The polymer shell is derived from the polymer in the solvent phase discussed above. Therefore, when using this method to produce particles, the composition of the polymer shell of the particles provided herein can be controlled by controlling the composition of the solvent phase in the disclosed method. In some embodiments, the composition of the polymer shell can be the same as the non-solvent component of the solvent phase. In some embodiments, the non-solvent component of the solvent phase is the same as the component of the polymer shell of the particle.

[0259] For example, the particles may include a solid polymer shell comprising one or more biodegradable polymers selected from poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(butylene succinate), poly(dioxanone) (PPDO), poly(hydroxybutyrate) (PHB), poly(butylene adipate-co-butylene terephthalate) (PBAT), chitosan, cellulose, hyaluronic acid, and blends thereof or copolymers thereof, such as PLGA. The L:G ratio of PLGA may be selected as described above. For example, the particles may include a solid polymer shell comprising PLGA having an average molecular weight of about 5 to about 100 kDa. The molecular weight may be selected or determined based on the desired release characteristics of the particles, as described above.

[0260] Typically, the particles produced according to the present invention have a polymer shell having a thickness of about 0.1 μm to about 100 μm, for example, a thickness of about 1 μm to about 50 μm, such as a thickness of about 3 μm to about 20 μm, for example, a thickness of about 5 μm to about 15 μm, such as a polymer shell having a thickness of about 10 μm. The thickness of the polymer shell is controllable as disclosed herein (e.g., by controlling the concentration of the polymer in the solvent phase and / or the flow rate used in the disclosed method, as described above), and allows the release characteristics of the particles to be precisely adjusted. The thickness of the polymer shell of the polymer can be easily determined, for example, by SEM or TEM, or by optical (e.g., fluorescence) microscopy.

[0261] Typically, the polymer shell of the particles of the present disclosure is highly uniform. Preferably, the thickness of the thinnest portion of the polymer shell is at least 70% of the thickness of the thickest portion of the polymer shell. More typically, the thickness of the thinnest portion of the polymer shell may be at least 90% of the thickness of the thickest portion of the polymer shell, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, at least 99.99% or at least 99.999% of the thickness of the thickest portion of the polymer shell. Preferably, the thickness of the thinnest portion of the polymer shell is indistinguishable from the thickness of the thickest portion of the polymer shell when measured, for example, by scanning electron microscopy (SEM) or optical microscopy (e.g., fluorescence microscopy).

[0262] It is within the ability of a person skilled in the art to vary the thickness of the shell depending on the application of the particle: this is an operating parameter that can be determined by a person skilled in the art.

[0263] The particles of the present disclosure are spherical, uniform and stable.

[0264] Typically, the average diameter of the present separated particles is about 5 μm to about 500 μm. More typically, the average diameter of the particles of the present disclosure is about 10 μm to about 250 μm, such as about 20 μm to about 150 μm, for example about 40 μm to about 100 μm, such as about 50 μm to about 80 μm, for example about 55 μm to about 70 μm.

[0265] Many methods are available for measuring particle diameter, such as SEM, dynamic light scattering, and optical (eg, fluorescence) microscopy.

[0266] The particles of the present disclosure are highly spherical. Those skilled in the art will appreciate that particles made by previously known methods typically deviate significantly from spherical shapes.

[0267] A mathematically perfect sphere can be considered to have a single diameter, regardless of the angle, direction, or plane of the vector defining the diameter. In practice, physical objects inevitably deviate from a mathematically perfect sphere. Therefore, the sphericity parameter of a particle of the present disclosure can be defined by comparing the smallest measurable diameter of the particle to the largest measurable diameter.

[0268] Preferably, the minimum diameter of the particle is at least 70% of the maximum diameter of the particle. More typically, the minimum diameter of the particle may be at least 90% of the maximum diameter of the particle, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, at least 99.99% or at least 99.999% of the maximum diameter of the particle. Preferably, the minimum diameter of the particle is indistinguishable from the maximum diameter of the particle when measured, for example, by scanning electron microscopy (SEM) or optical (e.g., fluorescence) microscopy.

[0269] Typically, the particles of the present disclosure are monodisperse. Typically, the particles of the present invention or compositions comprising the particles of the present invention have a polydispersity index (PDI) of 0.3 or less, preferably 0.2 or less, more preferably 0.1 or less.

[0270] The dispersibility of the disclosed particles in a sample can also be defined based on the coefficient of variation of the average diameter of the particles in the sample (where the coefficient of variation CV is defined as CV(%) = {standard deviation of diameter} / {average value of diameter}×100). Typically, the coefficient of variation is between about 0.1% and 20%, such as between about 1% and 15%, for example, about 4% to about 10%.

[0271] Typically, the present separated particles have a well-defined release profile. The release profile is generally sigmoidal in nature, as discussed in the examples. Thus, the aqueous core is released from the particle in three phases: (i) an initial plateau phase, in which little (if any) aqueous core is released from the particle, constituting an initial "delay" phase; (ii) a release phase in which the aqueous core is released; and (iii) a later plateau phase after at least a majority of the aqueous core in the sample has been released from the particle.

[0272] The time scales for each of these three phases - particularly phases (i) and (ii) - can be controlled by the user of the disclosed method: they are parameters that can be determined by selecting, for example, the size and composition of the aqueous phase corresponding to the aqueous nature of the particles produced; the thickness and composition of the polymer shell; and the nature of the therapeutic agent (if any) encapsulated in the core. It is within the ability of one skilled in the art to vary the composition of the particles to achieve the desired release kinetics. For example:

[0273] - Typically, the molecular weight of the polymer included in the solvent phase and therefore in the polymer shell of the resulting particle is increased to increase the time scale of the initial plateau, thereby delaying the release of the aqueous core from the polymer shell.

[0274] Typically, increasing the thickness of the polymer shell surprisingly appears to generally reduce the time frame of the initial plateau, thereby accelerating the release of the aqueous core from the polymer shell. Without being bound by theory, the inventors believe this may be due to the fact that thinner shells are less likely to degrade via autocatalysis.

[0275] Typically, changing the composition of the polymer included in the solvent phase, and therefore in the polymer shell of the resulting particle, changes the time frame of the initial plateau, thereby changing the rate of release of the aqueous core from the polymer shell. For example, when using a PLGA polymer as described herein, increasing the L:G ratio generally increases the time frame of the initial delayed plateau, thereby delaying the release of the aqueous core from the polymer shell.

[0276] Typically, the initial plateau region can last from about 1 day to about 1 year, such as from about 1 week to about 6 months, for example from about 2 weeks to about 4 months (e.g., from about 2 weeks to about 10, 11, 12, 13 or 14 weeks), for example, from about 2 weeks to about 2 months. Typically, the duration of the release phase is from about 1 hour to about 3 weeks, for example, from about 1 day to about 5 days, for example, from about 2 to 3 days. The length of the final plateau region is usually indefinite; it may be, for example, at least 1 day, at least 1 week; at least 1 month; at least 1 year or longer.

[0277] Pharmacy

[0278] As described above, the aqueous phase and thus the aqueous core of the provided particles include a pharmaceutical agent. Additional pharmaceutical agents may be present in a pharmaceutical composition comprising the particles and one or more additional components. A pharmaceutical agent may be present in the core of a particle and in a pharmaceutical composition comprising the particle. A pharmaceutical agent may be included in the core of a particle and a second pharmaceutical agent may be included in a pharmaceutical composition comprising the particle.

[0279] Examples of pharmaceutical agents that may be included in the aqueous phase and therefore included in the core of the disclosed particles and / or compositions comprising such particles include: proteins and other biomacromolecules, water-miscible drugs, virus-like particles, viruses, nano-sized particles, therapeutic bacteria, therapeutic cells, polysaccharides, and the like.

[0280] Examples of agents that may be included in the core of the disclosed particles and / or in compositions comprising such particles include: immunogenic agents; analgesics; antibiotics; antithrombotic agents (such as t-PA); antidepressants; anticancer agents, such as chemotherapeutic agents including combinations thereof (e.g., 5-fluorouracil, folinic acid (FA) or salts thereof, 6-mercaptopurine, cytarabine, gemcitabine, oxaliplatin, methotrexate, actinomycin-D, bleomycin, daunorubicin, doxorubicin, docetaxel, estramustine, paclitaxel, vinblastine, etoposide, irinotecan, teniposide, topotecan, prednisone, methylprednisolone, dexamethasone, and combinations of two or more of these chemotherapeutic agents); anti-epileptic drugs; anti-inflammatory drugs; antipsychotic drugs; antiviral drugs; sedatives; steroids; antidiabetic drugs; cardiovascular drugs, and drugs for pain control, treatment of skin disorders, and treatment of brain diseases. In some embodiments, the agent is an immunogenic agent. The immunogenic agent can be used as a vaccine, eg, a vaccine for preventing or treating (typically preventing) a disease or disorder herein.

[0281] The pharmaceutical agents used in the present disclosure may include or consist of therapeutic sugars such as D-mannose, L-fructose, D-galactose, N-acetyl-D-glucosamine, sialic acid, N-acetyl-D-mannosamine, ribitol. Glucan molecules are further examples of therapeutic sugars.

[0282] The particles of the present disclosure and compositions comprising such particles can also be used to deliver diagnostic agents including contrast agents and radioactive or tracer molecules. Specific examples of useful contrast agents are MRI contrast agents, such as GD-based MRI contrast agents or fluorescent contrast agents. Therefore, such agents can also be included in the aqueous phase and thus in the aqueous core.

[0283] The particles of the present disclosure and compositions comprising such particles are particularly suitable for the delivery of vaccines. The particles of the present disclosure and compositions comprising such particles are particularly suitable for the delivery of vaccines for the prevention (prophylactic treatment) of diseases described herein. Examples of suitable compositions and methods of administration of polypeptide fragments are provided in Esseku and Adeyeye (2011) and Van den Mooter G. (2006). Vaccine and immunotherapy composition preparation are commonly described in vaccine design ("The subunit and adjuvant approach" (Powell MF & Newman MJ (1995) Plenum Press New York).

[0284] The medicament used according to the present disclosure may include or be composed of a non-immunomodulatory polypeptide (or non-immunomodulatory protein), wherein the non-immunomodulatory polypeptide (or non-immunomodulatory protein) is a polypeptide (or protein) that does not cause an immune response when administered. Typically, such polypeptides or proteins are polypeptides or proteins naturally present in the subject. With respect to human subjects, an example of a non-immunomodulatory protein is human serum albumin. More generally, human blood proteins are examples of non-immunomodulatory proteins, including insulin, globulin, and hemoglobin.

[0285] The medicament used in the present disclosure may include an immunomodulatory polypeptide (or immunomodulatory protein) or be composed of an immunomodulatory polypeptide (or immunomodulatory protein). An immunomodulatory polypeptide (or immunomodulatory protein) is a polypeptide (or protein) that induces an immune response when administered to a subject. Typically, the subject is a human, and the protein is a protein that causes an immune response when administered. Typically, administration of an immunomodulatory polypeptide (or immunomodulatory protein) causes the subject to produce antibodies against the polypeptide (or protein), although alternative immune responses or antibodies may also be produced instead.

[0286] The immunomodulatory polypeptide can be, for example, an immune checkpoint regulator, an antibody, a vaccine antigen, an adjuvant or an anti-inflammatory polypeptide. Further examples of immunomodulatory polypeptides useful in the present invention include macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF, such as TNF-α), granulocyte macrophage colony stimulating factor (GM-CSF) and interferon.

[0287] Examples of suitable immune checkpoint regulators include: anti-PDL-1 (aPDL1); anti-PD1 (aPD1); anti-CTLA4 (aCTLA4); bispecific T cell engagers (BiTEs); cytokines; and chemokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, etc.).

[0288] Examples of suitable antibodies include: aPDL1; aPD1; aCTLA4; BiTE antibodies; anti-growth factor antibodies that also induce antibody-dependent cellular cytotoxicity (ADCC), such as anti-EGFR antibodies (e.g., cetuximab and Herceptin); antibodies with anti-inflammatory effects, such as anti-TNF (adalimumab, Humira); and JAK inhibitor antibodies.

[0289] Examples of suitable antigens include vaccine antigens. For example, at least one pathogenic antigen protein can be used. The pathogenic antigen protein can be a full-length protein or a fragment thereof. Examples of suitable antigen proteins include fragments of viral proteins or parasitic proteins. Antigens can be from pathogens selected from Cal09 (influenza), coronaviruses (e.g., beta-coronavirus or SARS-CoV-2), hepatitis B, Bordatella pertussis (pertussis), Streptococcus pneumoniae, meningococcus bacterium, human papillomavirus (HPV) or Plasmodium sporozoites (malaria parasites).

[0290] The antigen protein can be a viral spike protein, preferably a spike protein of a SARS-CoV-2 protein (e.g., SARS-CoV-2, S1 subunit protein (RBD)). On the other hand, the antigen is a circumsporozoite protein (CSP), which is a surface protein secreted by sporozoite parasites. On the other hand, the antigen is a HepB surface antigen protein (HBsAg). On the other hand, the antigen is associated with influenza virus and is selected from hemagglutinin and / or influenza neuraminidase. On the other hand, the antigen is filamentous hemagglutinin (pertussis). On the other hand, the antigen is a pneumococcal surface protein A (PspA). On the other hand, the antigen is selected from Neisseria adhesin A (NadA), Neisseria heparin binding antigen (NHBA) and / or factor H binding protein (fHbp). On the other hand, the antigen is an HPV-16E6 / E7 fusion protein. Fragments or genetically modified versions of any of these proteins can also be used.

[0291] The medicament used in the present disclosure may include a nucleic acid vaccine or be composed of a nucleic acid vaccine. In some embodiments, the nucleic acid vaccine is a DNA vaccine. In some embodiments, a DNA vaccine or a gene vaccine includes a plasmid having a promoter and suitable transcription and translation control elements and a nucleic acid sequence encoding one or more polypeptides of the present disclosure. In some embodiments, the plasmid also includes a sequence for enhancing, for example, expression levels, intracellular targeting, or proteasome processing. In some embodiments, a DNA vaccine includes a viral vector containing a nucleic acid sequence encoding one or more polypeptides. On the other hand, the composition disclosed herein includes one or more nucleic acids encoding peptides determined to be immunoreactive with a biological sample. For example, in some embodiments, the composition includes one or more nucleotide sequences encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more polypeptides, the polypeptide including a fragment of a T cell epitope capable of binding to at least three HLA class I molecules and / or at least three or four HLA class II molecules of a patient. In some embodiments, the peptide is derived from an antigen expressed in a cancer. In some embodiments, the DNA or genetic vaccine also encodes an immunomodulatory molecule to manipulate the resulting immune response, such as to enhance the efficacy of the vaccine, stimulate the immune system, or reduce immunosuppression. Strategies to enhance the immunogenicity of DNA or genetic vaccines include encoding a heterologous version of the antigen, fusing the antigen with a molecule that activates T cells or triggers cognate recognition, priming with a DNA vector followed by a boost with a viral vector, and utilizing immunomodulatory molecules.

[0292] The medicament used in the present disclosure may include RNA vaccines or be composed of RNA vaccines. In some embodiments, RNA is non-replicating mRNA or virus-derived self-amplifying RNA. In some embodiments, non-replicating mRNA encodes peptides disclosed herein and includes 5' and 3' untranslated regions (UTR). In some embodiments, virus-derived self-amplifying RNA not only encodes peptides disclosed herein, but also encodes viral replication mechanisms that can amplify RNA and enrich protein expression in cells. In some embodiments, RNA is directly introduced into an individual. In some embodiments, RNA is chemically synthesized or transcribed in vitro. In some embodiments, mRNA is produced from a linear DNA template using T7, T3 or Sp6 phage RNA polymerase, and the resulting product includes an open reading frame, flanking UTR, 5' cap and poly (A) tail encoding the peptide disclosed herein. In some embodiments, various versions of 5' caps are added during or after the transcription reaction using vaccinia virus capping enzymes or by incorporating synthetic caps or anti-reverse cap analogs. In some embodiments, poly (A) tails of optimal length are added to mRNA directly from a coding DNA template or by using poly (A) polymerase. The RNA may encode one or more peptides including a fragment that is a T cell epitope capable of binding to at least three HLA class I and / or at least three or four HLA class II molecules of the patient. In some embodiments, the fragment is derived from an antigen expressed in a cancer. In some embodiments, the RNA includes signals that enhance stability and translation. In some embodiments, the RNA also includes non-natural nucleotides that increase half-life or modified nucleosides that change the immune stimulation spectrum.

[0293] The medicament used in the present disclosure may include one or more cells, or be composed of one or more cells. The therapeutic administration of cells can be used for stem cell, progenitor cell or mature cell transplantation, differentiation and long-term replacement of damaged tissue. In this mode, multipotent or unipotent cells differentiate into specific cell types in the laboratory or after reaching the site of injury (by local or systemic administration). These cells are then integrated into the site of injury, replacing damaged tissue, thereby promoting the improvement of organ or tissue function. Examples in this regard are the use of cells to replace myocardial cells after myocardial infarction, promoting angiogenesis in ischemic limb disease, or producing cartilage in intervertebral disc degeneration. Further therapeutic uses of cells include the administration of cells with the ability to release soluble factors that can act in a paracrine or endocrine manner (such as cytokines, chemokines and growth factors). Examples in this regard include cells that secrete factors that promote angiogenesis, anti-inflammatory and anti-apoptotic factors. Adherent stromal cells or mature endothelial cells can be used to treat peripheral arterial disease and arteriovenous access complications. The cells administered by the disclosed method can be allogeneic, autologous or xenogeneic. Examples of suitable cell types include human embryonic stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, and differentiated or mature cells.

[0294] The medicament used in the present disclosure may include or be composed of a therapeutic microorganism, such as a bacterium. Examples include the use of tumor-homing bacteria that can target cancer cells in vivo. Some common tumor-homing bacteria include Salmonella, Clostridium, Bifidobacterium, Listeria, and Streptococcus. Typically, Salmonella kills tumor cells by uncontrolled bacterial proliferation, which can cause the rupture of cancer cells, and tumors colonized by Salmonella usually secrete anti-tumor IL-1β. S. Typhimurium flagellin is believed to increase the innate and adaptive immunity (nonspecific and specific defense mechanisms) of bacteria by stimulating NK cells (natural killer cells) to produce interferon-γ (IFN-γ), of which interferon-γ is an important cytokine (regulatory protein) for this immunity. Listeria is thought to suppress tumors through NADPH oxidase-mediated production of ROS (reactive oxygen species) (nicotinamide adenine dinucleotide phosphate oxidase), a cell signaling process that activates CD8+ T cells (cells that kill cancerous tissue) that target primary tumors. In addition, Clostridium, S. Typhimurium, and Listeria are thought to produce exotoxins (e.g., phospholipases, hemolysins, lipases) that disrupt tumor membrane structure and cellular function through apoptosis or autophagy, i.e., programmed cell death. Salmonella, Clostridium, and Listeria infections may also promote tumor elimination by increasing cytokines and chemokines that regulate the site of infection. Mycobacterium Bovis, also known as Bacillus Calmette-Guérin (BCG), is a proven treatment for bladder cancer.

[0295] An exemplary agent used in the present disclosure is R21. R21 is a new pre-erythrocytic malaria candidate vaccine that includes HBsAg fused to the C-terminus and central repeat region (Asn-Ala-Asn-Pro [NANP]) of CSP (circumsporozoite protein), which self-assembles into virus-like particles in yeast. R21 lacks the excess HBsAg found in other malaria candidate vaccines (such as RTS, S). R21 only includes the fusion protein portion, which is in contrast to RTS, S, which include 20% of the fusion protein and the remaining 80% are HBsAg monomers expressed separately, and therefore can reduce the coverage of CSP on the surface of virus-like particles. The R21 pre-erythrocytic malaria candidate vaccine was developed by the University of Oxford (Oxford, UK) and is currently produced by the Serum Institute of India (Pune, India).

[0296] Sometimes, particles of the present disclosure and / or compositions comprising such particles include an adjuvant. When an adjuvant is present, it can be present in the core of the particle (e.g., it can be present in the aqueous phase of the disclosed method) and / or it can be present in a pharmaceutical composition comprising the particle and one or more other components. An adjuvant can be present in the core of the particle and in a pharmaceutical composition comprising such particles. A first adjuvant can be included in the core of the particle and a second adjuvant can be included in a pharmaceutical composition comprising the particle. It will be apparent to those skilled in the art that when an adjuvant is present in the core of the particle, it will be present in the aqueous phase described above.

[0297] The term "adjuvant" as used herein refers to a substance used to enhance immunomodulation. Examples of adjuvants include: aluminum salts such as alum, aluminum hydroxide or aluminum phosphate; calcium salts (e.g., calcium hydroxide); iron salts; zinc salts; insoluble suspensions of acylated tyrosine; acylated sugars; cationically or anionically derivatized sugars; polyphosphazenes; biodegradable microspheres; lipid nanoparticles; monophosphoryl lipid A (MPL); lipopolysaccharides; lipid A derivatives (e.g., with reduced toxicity); 3-O-deacylated MPL [3D-MPL]; detergents such as quilA, saponin; QS21; Freund's Complete Adjuvant; Freund's Incomplete Adjuvant (Difeo Laboratories, Detroit, Mich); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (Smith Kline Beecham, Philadelphia, Pa.); CpG oligonucleotides; oils (such as squalene); mineral oils (such as paraffin oil); food-based oils, such as adjuvant 65 (extracted from peanut oil); bacterial products, such as inactivated bacteria selected from Bordetella pertussis and Mycobacterium bovis; toxoids; bioadhesives and mucoadhesives; microparticles; liposomes; polyoxyethylene ether preparations; polyoxyethylene ester preparations; muramyl peptides or imidazoquinolone compounds (such as imiquimod and its homologues), human immunomodulators suitable for use as adjuvants, including cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.); macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), granulocyte and macrophage colony stimulating factor (GM-CSF). An exemplary adjuvant for use herein is a liposome-based adjuvant having saponin QS21 and a TLR-4 agonist integrated into the liposome bilayer, which is suitable for use with the R21 vaccine composition described in Example 9.

[0298] Sometimes, the particles of the present disclosure and / or compositions comprising such particles include cytokines. When there is a cytokine, it can be present in the core of the particle (for example, it can be present in the aqueous phase of the disclosed method) and / or it can be present in a pharmaceutical composition comprising particles and one or more other components. The cytokine can be present in the core of the particle and in a pharmaceutical composition comprising such particles. A first cytokine can be included in the core of the particle, and a second cytokine can be included in a pharmaceutical composition comprising the particle. It is obvious to those skilled in the art that when the cytokine is present in the core of the particle, it will have been present in the aqueous phase described above. Cytokines are particularly useful in embodiments of the present disclosure, wherein the disclosed particles and compositions comprising them are used for vaccination, because the cytokine can strengthen the immunogenicity of the particles or compositions.

[0299] The cytokine may be selected from the group consisting of transforming growth factors (TGFs), such as but not limited to TGF-α and TGF-β; insulin-like growth factor-I and / or insulin-like growth factor-II; erythropoietin (EPO); osteoinductive factors; interferons, such as but not limited to interferon-α, interferon-β and interferon-γ; colony stimulating factors (CSFs), such as but not limited to macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and, granulocyte-CSF (G-CSF). In some embodiments, the cytokine is selected from the group consisting of: nerve growth factor, such as NGF-β; platelet growth factor; transforming growth factor (TGF), such as but not limited to TGF-α, TGF-β; insulin-like growth factor-I and insulin-like growth factor II; erythropoietin (EPO); osteoinductive factor; interferon (IFN), such as but not limited to IFN-α, IFN-β and IFN-γ; colony stimulating factor (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and, granulocyte-CSF (G-CSF); interleukins (IL), such as, but not limited to, IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18; LIF; kit-ligand or FLT-3; angiostatin; thrombospondin; endostatin; tumor necrosis factor (TNF) and LT.

[0300] Typically, the adjuvant or cytokine can be added in an amount of about 0.01 mg to about 10 mg per dose, preferably about 0.2 mg to about 5 mg per dose. Alternatively, the concentration of the adjuvant or cytokine can be about 0.01 to 50%, preferably about 2% to 30%.

[0301] Other therapeutic agents that may be included in the particles of the present disclosure and / or compositions comprising such particles; or other therapeutic agents administered in combination with the particles of the present disclosure and / or compositions comprising such particles include: checkpoint blockade therapy / checkpoint inhibitors, co-stimulatory antibodies, cytotoxic or non-cytotoxic chemotherapy and / or radiotherapy, targeted therapy or monoclonal antibody therapy. It has been shown that chemotherapy sensitizes tumors to be killed by tumor-specific cytotoxic T cells induced by vaccination (Ramakrishnan et al. J Clin Invest. 2010; 120(4): 1111-1124).Examples of chemotherapeutic agents include: alkylating agents, including nitrogen mustards such as mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-myosin), and chlorambucil; anthracyclines; epothilones; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), cemustine (methyl-CCNU), and streptozotocin (streptozotocin); triazenes such as decarbazine (DTIC; dimethyltriazolecarboxamide); ethyleneimines / methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulfonates such as busulfan; antimetabolites, including folic acid analogs; drugs, such as methotrexate (AMT); alkylating agents, antimetabolites, pyrimidine analogs such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (arabinosylcytosine); purine analogs and related inhibitors, such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG) and pentostatin (2'-deoxyformycin); epipodophyllotoxins; enzymes, such as L-asparaginase; biological response modifiers, such as IFNα, IL-2, G-CSF and GM-CSF; platinum coordination complexes, such as cisplatin (cis-DDP), oxaliplatin and carboplatin; anthracene dihydrotestosterone; ketones, such as mitoxantrone and anthracyclines; substituted ureas, such as hydroxyurea; methylhydrazine derivatives, including procarbazine (N-methylhydrazine, MIH) and procarbazine; adrenocortical suppressants, such as mitotane (o, p'-DDD) and aminoglutethimide; paclitaxel and analogs / derivatives; hormones / hormone therapies and agonists / antagonists, including adrenocortical steroid antagonists, such as prednisone and its equivalents, dexamethasone and aminoglutethimide, progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate), estrogens, (such as diethylstilbestrol and ethinyl estradiol equivalents), antiestrogens (such as tamoxifen), androgens (including testosterone propionate and flumethoxetine) testosterone / equivalents), antiandrogens (such as flutamide), gonadotropin-releasing hormone analogs and leuprolide and nonsteroidal antiandrogens (such as flutamide); natural products, including vinca alkaloids (such as vinblastine (VLB) and vincristine), epipodophyllotoxins (such as etoposide and teniposide), antibiotics (such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin (doxorubicin), bleomycin, primycin (miltomycin) and mitomycin (mitomycin C), enzymes (such as L-asparaginase), and biological response modifiers (such as interferon alpha).

[0302] Composition

[0303] The particles of the present disclosure may be provided in the form of a pharmaceutical composition comprising the particles of the present disclosure and one or more pharmaceutically acceptable excipients, diluents or adjuvants.

[0304] Preferably the pharmaceutical compositions are sterile and pyrogen-free.

[0305] When the pharmaceutical compositions provided herein include one or more optically active components (e.g., one or more optically active agents), these components (e.g., agents) are generally in the form of substantially pure optical isomers. For the avoidance of doubt, the components may be administered in the form of solvates.

[0306] Typically, the composition contains up to 85 wt% of the disclosed particles. More typically, it contains up to 50 wt% of the disclosed particles, such as up to 40 wt%, up to 30 wt%, up to 20 wt%, or up to 10 wt% of the disclosed particles.

[0307] In some embodiments, the composition includes a physiologically acceptable medium in which the particles can be administered to a subject. The medium may include water or be composed of water (e.g., deionized water and / or ultrapure water). The medium may have a pH of about 4 to about 10. More generally, the pH of the medium is about 5 to about 9, such as about 6 to about 8. The medium may include one or more buffer salts. The buffer salts are usually present with one or more additional pharmaceutically acceptable salts so that the resulting composition is isotonic, such as isotonic with human plasma. Pharmaceutically acceptable buffers include phosphate / phosphoric acid, citrate / citric acid, acetate / acetic acid, histidine, lactate / lactic acid, tromethamine, gluconic acid / gluconate, aspartic acid / aspartate, tartaric acid / tartrate, succinic acid / succinate, malic acid / malate, fumaric acid / fumarate, and α-ketoglutaric acid / α-ketoglutarate. Most commonly, the buffer salts are selected from phosphate, citrate, and acetate. The preferred medium is phosphate buffered saline (PBS). In some embodiments, the PBS includes NaCl and / or KCl, and is buffered to about 7.4 using a phosphate buffer. For example, the PBS may include 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4.

[0308] As mentioned above, the particles of the present disclosure can be used to deliver any suitable therapeutic agent. Obviously, one or more therapeutic agents are generally included in the aqueous core of the particles disclosed herein. When formulated into a composition including other components in addition to the particles, one or more additional agents can be included in the medium present in the particles. Any agent disclosed herein can be used in the aqueous core of the particles and / or in the medium of the composition including such particles.

[0309] The particles of the present disclosure and compositions comprising them can be used to deliver vaccines. Accordingly, provided herein are vaccine compositions comprising a plurality of particles disclosed herein, wherein the particles comprise an agent as a first immunogenic agent.

[0310] The delayed release nature of the particles means that they are suitable for use in prime / boost vaccinations.

[0311] In this context, those skilled in the art will appreciate that, as described above, the goal of vaccination is to induce long-term immune memory to mediate protection against infection, or at least to prevent disease upon exposure to a pathogen. Most vaccine strategies require multiple immunizations to induce effective protection.

[0312] Many vaccines require multiple doses to generate long-term protective immunity at high frequencies and ensure strong individual and herd immunity. Repeated immunogenic stimulation not only increases the strength and durability of adaptive immunity, but also affects its quality.

[0313] The term "priming dose" can be used interchangeably with "boosting dose", especially when multiple vaccine injections are close in time. Typically, the first vaccine dose activates naive T cells and B cells, which undergo a program of proliferation, contraction, and differentiation to develop into primary memory T cells. Once the second dose of the vaccine is injected, when the main effector response begins to contract, it can be called a boost. It has been shown that repeated administration of exactly the same vaccine (called homologous priming / boosting) is very effective in increasing humoral responses. However, they appear to be relatively inefficient in enhancing cellular immunity, probably because previous immunity to the vaccine tends to impair robust Ag presentation and the production of appropriate inflammatory signals for T cells. In contrast, the sequential administration of vaccines using different Ag delivery systems is called heterologous priming / boosting. In preclinical studies and clinical trials, it has been shown to effectively produce high levels of memory T cells.

[0314] It is known that several vaccine parameters influence the adaptive immune response, including, among others, the number of immunizations, the delay between immunizations, and the order of delivery of different recombinant vaccine vectors. The delayed release properties of the disclosed particles mean that compositions comprising such particles are ideal for delivering prime / boost benefits in a single administration, where the prime dose comes from the therapeutic agent in the bulk composition and the boost dose comes from the therapeutic agent (same or different) in the aqueous core of the particle.

[0315] Therefore, there is further provided a prime / boost vaccine composition comprising:

[0316] (i) a plurality of particles disclosed herein, wherein the particles comprise an agent as a first immunogenic agent; and

[0317] (ii) a second immunogenic agent;

[0318] Wherein the first immunogenic agent and the second immunogenic agent are the same or different. When the two immunogenic agents are the same, the prime / boost vaccine composition can be referred to as a homologous prime / boost vaccine composition. When the two immunogenic agents are different, the prime / boost vaccine composition can be referred to as a heterologous prime / boost vaccine composition.

[0319] The particles of the present disclosure and compositions comprising such particles can be administered in a variety of dosage forms. Therefore, they can be administered orally, for example in the form of tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. They can also be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrasternal, transdermally, or by infusion techniques. The compound, composition or combination can also be administered in the form of a suppository. Preferably, the compound, composition or combination is administered parenterally, particularly by intravenous administration.

[0320] The particles of the present disclosure and compositions comprising such particles are typically formulated for administration together with a pharmaceutically acceptable carrier or diluent.

[0321] For example, solid oral forms may contain: diluents, such as lactose, dextrose, sucrose, cellulose, corn starch or potato starch; lubricants, such as silicon dioxide, talc, stearic acid, magnesium or calcium stearate, and / or polyethylene glycol; binders, such as starch, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinylpyrrolidone; deagglomerants, such as starch, alginic acid, alginates or sodium starch glycolate; effervescent mixtures; dyes; sweeteners; wetting agents, such as lecithin, polysorbate, lauryl sulfate / ester; and non-toxic and pharmacologically inactive substances commonly used in pharmaceutical formulations. Such pharmaceutical preparations can be manufactured in a known manner, for example, by mixing, granulating, tableting, sugar coating or film coating processes.

[0322] The particles of the present disclosure and compositions comprising such particles can be formulated for pulmonary administration. For example, the particles of the present disclosure and compositions comprising such particles can be formulated into solutions or suspensions for inhalation (atomization) administration. The particles of the present disclosure and compositions comprising such particles can be administered by a metered dose inhaler (MDI) or a nebulizer (such as an electronic nebulizer or a jet nebulizer). Alternatively, the particles of the present disclosure and compositions comprising such particles can be formulated into powdered medicaments for inhalation administration; such preparations can be administered by a dry powder inhaler (DPI).

[0323] Liquid dispersions for oral administration may be syrups, emulsions and suspensions. Syrups may contain sucrose or sucrose containing glycerol and / or mannitol and / or sorbitol as carriers.

[0324] Suspensions and emulsions may contain carriers such as natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol. Suspensions or solutions for intramuscular injection may contain, in addition to the active compound, a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycol (such as propylene glycol), and, if desired, an appropriate amount of lidocaine hydrochloride.

[0325] Solutions for injection may contain, for example, sterile water as a carrier, or preferably, they may be in the form of sterile, aqueous, isotonic saline solutions. Compositions for injection (e.g., intravenous administration) may contain excipients for increasing the solubility of the component compounds. Suitable excipients include cyclodextrins, such as sulfobutyl beta-cyclodextrin sodium (captisol). Pharmaceutical compositions suitable for, for example, transdermal administration by needle-free injection may also be used.

[0326] Treatment: Effect

[0327] The particles of the present disclosure and compositions comprising such particles are particularly advantageous in medical systems.

[0328] The particles of the present disclosure and compositions comprising such particles are useful in therapy. Thus, provided herein are particles of the present disclosure and compositions comprising such particles for use in medicine. The present invention also provides particles of the present disclosure and compositions comprising such particles for use in treating the human or animal body. The present invention also provides particles of the present disclosure and compositions comprising such particles for use in preventing disease in the human or animal body.

[0329] The particles of the present disclosure and compositions comprising such particles can be administered in combination with one or more additional pharmaceutically active agents described herein. The active agents can each be provided in the form of a single formulation, or one or more of them can be formulated separately. In the case of separate formulations, two or more agents can be administered simultaneously or separately. Separately formulated active agents can be provided in the form of a kit, optionally together with instructions for their administration.

[0330] In some cases, the particles of the present disclosure and compositions comprising such particles are used in a method of vaccination or a method of providing immunotherapy. As used herein, "immunotherapy" is to treat or prevent a disease or condition by inducing or enhancing an immune response in an individual. In certain embodiments, immunotherapy refers to a therapy comprising administering one or more drugs to an individual to elicit a T cell response. The T cell response may be a cytotoxic T cell response to cells that display tumor associated antigens (TAA), tumor specific antigens (TSA), or cancer testis antigens (CTA) on their cell surfaces. In another embodiment, immunotherapy refers to a therapy that includes administering or expressing a polypeptide to an individual to elicit a T helper response, thereby providing costimulation for cytotoxic T cells that recognize and kill diseased cells. In another embodiment, immunotherapy refers to a therapy that includes administering one or more drugs to an individual to reactivate existing T cells to kill target cells.

[0331] In some cases, immunotherapy can be used to treat tumors. In other cases, immunotherapy can be used to treat diseases or disorders based on intracellular pathogens. In some cases, the present disclosure relates to the treatment of cancer or any specific type of cancer disclosed herein. In some other cases, the present disclosure relates to the treatment of viral, bacterial, fungal or parasitic infections, or any other disease or condition that can be treated by immunotherapy.

[0332] The particles of the present disclosure can be used to deliver vaccine therapeutics to address (e.g., prevent or treat, typically prevent) any suitable disease. Exemplary diseases that can be addressed by vaccines include cholera, COVID-19, dengue fever, diphtheria, Ebola, Haemophilus influenzae type b, hepatitis A, hepatitis B, hepatitis E, human papillomavirus infection, influenza, Japanese encephalitis, malaria, measles, meningococcal disease, mumps, pneumococcal disease, pertussis, poliomyelitis, rabies, rotavirus gastroenteritis, rubella, tetanus, tick-borne encephalitis, tuberculosis, typhoid fever, chickenpox, yellow fever, Herpes Zoster, Bordetella, canine distemper, canine influenza, canine parvovirus, chlamydia, feline calicivirus, feline distemper, feline leukemia, feline viral rhinotracheitis, leptospirosis, and Lyme disease. In some preferred embodiments, the particles of the present disclosure may be used to deliver vaccines for the prevention or treatment of malaria.

[0333] Therefore, provided herein are particles of the present disclosure and compositions comprising such particles, which are used to treat or prevent diseases in subjects in need thereof, wherein the use comprises administering the particles or the compositions to the subjects, thereby inducing an immunogenic response in the subjects. Also provided are methods of inducing an immunogenic response in subjects in need thereof, the methods comprising administering to the subjects a therapeutically effective amount of one or more particles of the present disclosure and / or one or more compositions containing such particles. Further provided are the uses of particles of the present disclosure and compositions comprising such particles in the preparation of a medicament for inducing an immunogenic response in a subject. In some embodiments, the particles and compositions are used to treat or prevent diseases described herein. In some embodiments, the particles and compositions are used to treat or prevent diseases described herein, wherein the use comprises administering the particles and compositions in the form of a primary immunization / boost vaccine. In some embodiments, the particles and compositions are used to prevent diseases described herein, wherein the use comprises administering the particles and compositions in the form of a primary immunization / boost vaccine. In some embodiments, the disease is malaria.

[0334] Further provided are particles of the present disclosure and compositions comprising such particles for use in methods of vaccination, the use comprising administering an effective amount of the particles and / or the composition to a subject in need thereof. Also provided are methods of vaccination, the method comprising administering an effective amount of one or more particles of the present disclosure and / or a composition comprising such particles to a subject in need thereof. Further provided are uses of particles of the present disclosure and / or compositions comprising such particles in the preparation of vaccines or in the preparation of medicaments for administration as vaccines. In some embodiments, the effective amount is a therapeutically effective amount of the particles and / or the composition for treating pathological conditions, such as conditions disclosed herein. In some embodiments, the effective amount is a prophylactic effective amount of the particles and / or the composition for preventing pathological conditions, such as conditions disclosed herein.

[0335] As described above, compositions comprising particles of the present disclosure are ideally suitable for primary / boost vaccination. Therefore, such compositions are further provided: comprising (i) particles of the present disclosure, wherein the particles comprise a first immunogenic agent; and (ii) a second immunogenic agent; the composition is used for a method of primary / boost vaccination, the use comprising administering an effective amount of the composition to a subject in need. In some embodiments, the particles comprise a therapeutic agent suitable for use as a vaccine provided herein, for example, a therapeutic agent suitable for use as a vaccine for the treatment or prevention (usually prevention) of malaria. In some embodiments, the therapeutic agent comprises a vaccine antigen. In some embodiments, the therapeutic agent comprises a pre-erythrocytic malaria vaccine.

[0336] Also provided are methods of prime / boost vaccination comprising administering to a subject in need thereof an effective amount of a composition comprising (i) a particle of the disclosure, wherein the particle comprises a first immunogenic agent, and (ii) a second immunogenic agent.

[0337] Further provided is the use of a composition comprising (i) a particle of the present disclosure, wherein the particle comprises a first immunogenic agent, and (ii) a second immunogenic agent, in the preparation of a medicament for administration as a prime / boost vaccine. In such uses and methods, the first immunogenic agent and the second immunogenic agent may be the same or different. When the two immunogenic agents are the same, the prime / boost vaccination method may be referred to as a homologous prime / boost vaccination. When the two immunogenic agents are different, the prime / boost vaccination method may be referred to as a heterologous prime / boost vaccination.

[0338] Those skilled in the art will appreciate that the term "prime / boost" as used herein does not necessarily require multiple doses of the particles provided herein, although this is not excluded. On the contrary, the delayed release properties of the particles provided herein mean that a prime / boost regimen can be provided by administering a single composition of particles of the present invention comprising an initial therapeutic agent and a secondary therapeutic agent (which may be the same or different from the initial therapeutic agent). The initial therapeutic agent provides an initial therapeutic effect ("prime"), and the secondary therapeutic agent provides a secondary therapeutic effect ("boost") after being released from the particles. For example, a therapeutic agent, such as a vaccine agent, can be provided in an aqueous suspension, and another vaccine agent that is the same or different can be provided in the particles suspended in the aqueous suspension. Example 9 describes such a composition and its therapeutic use, and constitutes a single-dose prime / boost strategy for effective treatment. Example 9 describes an exemplary embodiment of using the R21 vaccine for malaria, but the present invention is not limited accordingly, and those skilled in the art will appreciate that other vaccines can be used for the same or other diseases according to the requirements of the user of the present invention.

[0339] Further provided are methods for using the particles of the present disclosure and compositions comprising such particles for immunotherapy, the use comprising administering an effective amount of the particles and / or the composition to a subject in need thereof. Also provided are methods for immunotherapy, the method comprising administering an effective amount of one or more particles of the present disclosure and / or compositions comprising such particles to a subject in need thereof. Further provided are uses of the particles of the present disclosure and / or compositions comprising such particles in the preparation of immunotherapy drugs. In some embodiments, the effective amount is a therapeutically effective amount of the particles and / or the composition for treating a pathological condition, such as a condition disclosed herein. In some embodiments, the effective amount is a preventive effective amount of the particles and / or the composition for preventing a pathological condition, such as a condition disclosed herein.

[0340] In one aspect, the subject to be treated is a mammal, particularly a human. However, it may also be a non-human. Preferred non-human animals include, but are not limited to, primates, such as apes or monkeys; commercially farmed animals, such as horses, cattle, sheep or pigs; and pets, such as dogs, cats, mice, rats, guinea pigs, ferrets, gerbils or hamsters. The subject may be any animal that can be infected by bacteria.

[0341] The disclosed particles and / or compositions can be administered to a subject to prevent the onset or recurrence of one or more symptoms of a pathological condition. This is a preventive measure. In this embodiment, the subject can be asymptomatic. A preventive effective amount of the particles and / or compositions is administered to such a subject. A preventive effective amount is an amount that prevents the onset of one or more symptoms of a pathological condition.

[0342] The disclosed particles and / or compositions can be administered to a subject to treat one or more symptoms of a pathological condition. In this embodiment, the subject is typically symptomatic. A therapeutically effective amount of the particles and / or compositions is administered to such a subject. A therapeutically effective amount is an amount that effectively improves one or more symptoms of the disease.

[0343] A therapeutically or prophylactically effective amount of the compound of the present invention is administered to the subject. The dosage can be determined according to various parameters, in particular according to the compound used; the age, weight and condition of the subject to be treated; the route of administration; and the desired regimen. The doctor will be able to determine the route of administration and dosage required for any particular subject. Depending on the activity of the specific agent, the age, weight and condition of the subject to be treated, the type and severity of the disease, and the frequency and route of administration, a typical daily dose is about 0.01 to 100 mg per kg of body weight, preferably about 0.1 mg to 50 mg, for example, about 1 to 10 mg. Preferably, the daily dose level is 70 mg to 3.5 g. Sometimes, depending on the activity of the specific agent, the age, weight and condition of the subject to be treated, the type and severity of the disease, and the frequency and route of administration, a higher dose is required, such as about 0.01 to about 250 mg per kg of body weight, for example, about 0.1 mg / kg to about 200 mg / kg, for example, about 1 / kg of body weight to about 150 mg / kg of body weight. The preferred daily dose level can be, for example, 70 mg to 8 g. Higher doses may be particularly appropriate if the medicament is to be administered to a subject multiple times per day, for example 2, 3 or 4 times per day, for example 4 times per day. A suitable daily dose may be 70 mg to 8 g per day, administered in 2, 3 or 4 separate doses.

[0344] Other aspects of the present disclosure

[0345] Also provided herein are aqueous core-polymer shell particles that can be obtained by performing the methods disclosed herein. In some embodiments, the particles are particles described in more detail herein; for example, the particles can include an aqueous core described herein; can include a polymer shell described herein; can be included in a composition described herein; and / or can be used in a medicament described in more detail herein.

[0346] Also provided herein is a population of aqueous core-polymer shell particles. The particles in the population are particularly characterized by homogeneity. Therefore, in some embodiments, the present disclosure provides a homogeneous population comprising a plurality of aqueous core-polymer shell particles; wherein at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9% or at least 99.99% of the particles in the population, preferably at least 99.999% or to 99.9999% of the particles in the population are characterized in that they include:

[0347] a single, spherical, aqueous core having a volume average diameter of about 1 μm to about 300 μm (e.g., about 3 μm to about 150 μm, for example about 10 μm to about 100 μm, such as about 30 μm to about 70 μm, for example about 40 μm to about 55 μm), and a minimum diameter of the aqueous core that is at least 70% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, at least 99.99% or at least 99.999%) of the maximum diameter of the aqueous core; and

[0348] - A biodegradable polymer shell having a thickness of about 0.1 μm to about 100 μm (e.g., about 1 μm to about 50 μm, such as about 3 μm to about 20 μm, for example, about 5 to about 15 μm, such as about 10 μm), and the thickness of the thinnest part of the polymer shell is at least 70% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.9%, at least 99.99% or at least 99.999%) of the thickness of the thickest part of the polymer shell.

[0349] In some embodiments, each of the particles in the population comprises: a buffered aqueous core comprising one or more pharmaceutical agents as defined herein; and / or a biodegradable polymer in the shell comprising poly(lactic-co-glycolic acid) (PLGA) having an average molecular weight of about 1 kDa to about 250 kDa (e.g., about 5 kDa to about 100 kDa, such as about 10 kDa to about 50 kDa) and comprising a lactic acid to glycolic acid ratio (L:G ratio) of 60:40 to 90:10 (e.g., about 70:30 to about 80:20, such as about 75:25).

[0350] As described above, previously known methods are generally unable to provide such homogeneous populations because the methods used to produce known particles cannot achieve such high precision and uniformity. Without being bound by theory, the inventors believe that the improvements in homogeneity of the particle populations achievable according to the present disclosure can have a beneficial effect in providing a precise and controllable release of a medicament from the particle population, for example, when administered in vivo as described herein. Therefore, the particle populations provided herein can be used in the methods of treatment described herein, such as for treating or preventing one or more pathological conditions described herein.

[0351] It is to be understood that, although specific embodiments, specific configurations, and materials and / or molecules according to the inventive method have been discussed herein, various changes or modifications in form and detail may be made without departing from the scope and spirit of the invention. The following examples are provided to better illustrate specific embodiments, and they should not be considered as limitations of the present application. The present application is only limited by the claims.

[0352] Example

[0353] Example 1

[0354] This example describes the production of particles according to the present disclosure.

[0355] The microfluidic chip design described in this paper produced Figure 8 Particles shown. The widths of the aqueous phase, solvent phase, and aqueous extraction phase inlets were 20, 40, and 80 μm, respectively. The height of the channel was 50 μm before the aqueous phase extraction intersection and 100 μm thereafter. The design was made using CAD software (AutoCAD, Autodesk), and a positive mold was created from the design using soft lithography provided by an external supplier (Micruxtechnologies). In brief, a negative photoresist was spin-coated on a silicon wafer, baked, and selectively exposed to UV using a mask including the corresponding chip design. The wafer was then developed by chemical treatment to keep the positive structure of the chip on it. After further baking, the mold was siliconized with trimethylchlorosilane to reduce the viscosity of polydimethylsiloxane (PDMS).

[0356] Once fabricated, a mixture of PDMS prepolymer and curing agent (Sylgard 184) in a 10:1 weight ratio was poured onto the positive mold, and the PDMS was then cured overnight in an oven at 60°C before being peeled off the mold, thereby engraving the design within the PDMS block (negative printing). Inlets and outlets were punched using an 18G blunt needle (BD) and the chip was sealed with another piece of PDMS, using an O2 plasma cleaner (HPT-200, Henniker) as surface activation. Immediately after bonding, a coating solution of 1% polyvinyl alcohol (PVA) (87-90% hydrolyzed, 30-70 kDa molecular weight - MW) was injected through the extraction solvent inlet, coating the second intersection of the design and downstream, spatially restricted by the solvent and air injection of the aqueous core inlet. The coating solution was allowed to flow for 5 minutes, and the chip was then dried with air and cured on a hot plate at 115°C. This cycle was repeated 3 times to ensure successful hydrophilic coating in the correct channels. The chip was then kept in an oven at 60 °C for 2 days before being used for microfluidic production.

[0357] Using the chip fabricated as described above, Figure 8 Microfluidic production of the particles shown. Fluids were injected into the chip using a pressure controller (OB1 controller, Elveflow) and production was monitored using an inverted microscope (DMi8, Leica) and a high-speed camera (Nac MEMRECAM). The aqueous core, solvent and extraction streams were injected at initial pressures of 50 mbar, 120 mbar and 280 mbar, respectively, and then visually adjusted to achieve the correct production volume and emulsion size. The composition of each stream was as follows:

[0358] Internal aqueous core: 50 mg / mL Dextran TRITC (40 kDa MW) in PBS (pH 7.4, 1X)

[0359] • Solvent with polymer: 17.5% w / v PLGA (7-17 kDa MW, 50:50 L:G) in DMC

[0360] Aqueous extraction phase: 3% w / v PVA (9-10 kDA, 80% hydrolyzed) in DPBS

[0361] (TRICT = tetramethylrhodamine; PBS = phosphate-buffered saline; DMC = dimethyl carbonate; DPBS = Dulbecco's phosphate-buffered saline)

[0362] The resulting dual solution was collected in 10 mL of aqueous extraction phase in a glass Petri dish ( Figure 8 , left). The shell was hardened by extracting the solvent in a large amount of aqueous extraction phase in a glass bottle and rotating at room temperature for 24 hours to obtain solid PLGA shell, liquid dextran-loaded aqueous core particles ( Figure 8 , right). Images were taken using an inverted fluorescence microscope (Eclipse TiE, Nikon), and the bright field images were overlaid with the signals from the bright field images and TRITC imaging.

[0363] The resulting particle products exhibit high monodispersity and regularity in their core-shell geometry, suitable for medical applications such as delayed delivery of pharmaceutical ingredients.

[0364] Example 2

[0365] This example demonstrates that the release characteristics of particles produced according to the present disclosure can be controlled by controlling the polymer used to encapsulate the aqueous core.

[0366] Particles with polymer shells of varying molecular weight (MW) were produced as described in Example 1, varying the MW of the polymer simply by adjusting the flow composition of the solvent phase. The following solvent phase composition was used:

[0367] 17.5% w / v PLGA (7-17 kDa MW, 50:50 L:G) in DMC ( Fig.14 , the red data in the original color image),

[0368] 12.5% ​​w / v PLGA (24-38 kDa MW, 50:50 L:G) in DMC ( Fig.14 , blue data in the original color image),

[0369] 10.5% w / v PLGA (38-54 kDa MW, 50:50 L:G) in DMC ( Fig.14 , yellow data in the original color image),

[0370] 8.5% w / v PLGA (54-69 kDa MW, 50:50 L:G) in DMC ( Fig.14 , the data in the original color image is green),

[0371] The other compositions of the aqueous core and extraction phases remained unchanged.

[0372] For each formulation, batches of approximately 100,000 particles (one batch per MW) were made.

[0373] An in vitro model was used to evaluate the release properties of these different types of particle batches. Therefore, for each MW formulation, the particles were divided into 3 replicates of equal amounts of particles and suspended in 2 mL PBS (pH 7.4, 1X) in LoBind Eppendorf tubes. The tubes were incubated at 37°C in the dark with constant rotation at 20 rpm.

[0374] At regular time intervals, the particles were gently sedimented by centrifugation (30 rcf, 1 min), 1 mL of supernatant was collected, and the amount of dextran-TRITC released was measured spectroscopically (520 / 560 nm ex / em, Fluostar Omega, BMG Labtech). The supernatant was replenished with fresh PBS. Release curves were prepared by normalizing the amount of dextran-TRITC released from the aqueous core at a given time point to the total amount initially encapsulated. The cumulative curves for each formulation are shown in Table 1. Fig.14 Shown (mean of 3 replicates, error bars indicate standard deviation).

[0375] like Fig.14 As shown, the in vitro release delay of batches of particles is affected by the MW of the polymer constituting the particle shell. Higher MW increases the delay before the core contents (in this example, dextran-TRITC) are released. Therefore, the MW is increased from 7-17 kDa ( Fig.14 , red) increased to 54-69 kDa ( Fig.14 , green), would cause an increase in latency of approximately 2.5 weeks.

[0376] Thus, this example demonstrates that the release characteristics of particles produced according to the present disclosure can be controlled by controlling the nature of the polymer used to encapsulate the aqueous core.

[0377] Example 3

[0378] This example demonstrates that the release characteristics of particles produced according to the present disclosure can be controlled by controlling the thickness of the polymer shell used to encapsulate the aqueous core.

[0379] Particles with different shell thicknesses were produced as described in Example 1, with only the injection pressure adjusted to control the relative size of the droplets. The solvent phase was the same as in Example 1 (17.5% w / v PLGA 7-17 kDa MW in DMC, 50:50 L:G). A core loaded with dextran-TRITC (40 kDa MW) served as a fluorescent payload model.

[0380] The injection pressure of the aqueous phase was adjusted from 40 to 50 mbar, and the injection pressure of the solvent phase was adjusted from 100 to 120 mbar, and three batches of particles with different shell thicknesses were prepared.

[0381] Shell thickness was measured by fluorescence imaging (36 images, Eclipse TiE, Nikon) and processed using a custom MATLAB script to measure the average shell thickness for each batch.

[0382] Release from each batch was assessed in vitro according to the protocol described in Example 2 and the released fluorescent dextran-TRITC was sampled periodically.

[0383] like Fig.15 As shown, the particles with shell thickness of 5.6 and 7.2 μm ( Fig.15 , red and orange in the original color image) were released simultaneously within 16 to 24 days, while the thin-shell particles with a shell thickness of 3.5 μm ( Fig.15 , yellow in the original color image) exhibited a small amount of release in about the same time, but held the load longer. Without being bound by theory, the inventors believe that this difference may be due to the fact that particles with thicker shells undergo higher autocatalytic degradation, while thin shell particles do not hold the degradation byproducts long enough to achieve a high degree of autocatalytic degradation and therefore take longer to release their payload.

[0384] Thus, this example demonstrates that the release characteristics of particles produced according to the present disclosure can be controlled by controlling the thickness of the polymer shell used to encapsulate the aqueous core.

[0385] Example 4

[0386] This example demonstrates that the release characteristics of particles produced according to the present disclosure, wherein the polymer is PLGA, can be controlled by controlling the lactic acid to glycolic acid (L:G ratio) content of the PLGA polymer.

[0387] Particles with PLGA polymer shells having different lactic acid to glycolic acid ratios (L:G) were produced as described in Example 1, simply adjusting the solvent phase flow composition to vary the L:G of the polymer. The following solvent phase compositions were used:

[0388] 12.5% ​​w / v PLGA (24-38 kDA MW, 50:50 L:G) in DMC ( Fig.14 , light blue),

[0389] · 12.5% ​​w / v PLGA (24-38 kDA MW, 75:25 L:G) in DMC ( Fig.14 , dark blue),

[0390] The other compositions of the aqueous core and extraction phases remained unchanged.

[0391] For each formulation, batches of approximately 100,000 particles (one batch per MW) were made.

[0392] Release from each batch was assessed in vitro according to the protocol described in Example 2 and the released fluorescent dextran-TRITC was sampled periodically.

[0393] like Fig.16As shown, the in vitro release delay of batches of particles is affected by the L:G ratio of the polymer constituting the particle shell. A higher L:G ratio increases the delay before the core contents (in this example, dextran-TRITC) are released. Therefore, while keeping the MW constant at 24-38 kDa, the L:G ratio was increased from 50:50 ( Fig.14 , light blue) increased to 75:25 ( Fig.14 , dark blue), would significantly increase the delay by about 8 weeks.

[0394] Thus, this example demonstrates that the release characteristics of particles produced according to the present disclosure can be controlled by controlling the nature of the polymer used to encapsulate the aqueous core (eg, by controlling the L:G ratio of the PLGA polymer).

[0395] Example 5

[0396] This example demonstrates that the thickness of particles produced by the present disclosure can be controlled by controlling the injection pressure of the aqueous phase and the solvent phase.

[0397] Particles with different shell thicknesses were produced as described in Examples 1 and 3, with the injection pressure adjusted to control the relative size of the droplets. The solvent phase was the same as in Example 1 (17.5% w / v PLGA 7-17 kDa MW in DMC, 50:50 L:G). A core loaded with dextran-TRITC (40 kDa MW) served as a fluorescent payload model.

[0398] The injection pressures for the aqueous core and solvent phases were adjusted to approximately 50 mbar and 100 mbar, respectively ( Fig.17A ), or the injection pressures for the aqueous core and solvent phases were adjusted to 40 mbar and 120 mbar, respectively ( Fig. 17B ). The extraction phase injection pressure was maintained at 280 mbar. Manual fine-tuning of the injection pressure was performed due to visual monitoring using a high-speed camera.

[0399] As shown in Figure 17, changing the injection pressure ratio of the aqueous core and solvent phases changes the thickness of the particles. By increasing the pressure ratio of the aqueous phase to the solvent, and thus increasing the flow rate ratio of the aqueous phase to the solvent, particles with thinner shells ( Fig.17A In contrast, when the aqueous phase to solvent pressure ratio is low, particles with thicker shells are produced ( Fig. 17B ).

[0400] Thus, this example demonstrates that by controlling the injection pressures of the aqueous and solvent phases in the disclosed process, and thus the flow rate ratio, the release characteristics of the particles produced by the disclosed process can be controlled.

[0401] Example 6

[0402] This example demonstrates that the immune response generated by in vivo administration of an immunogenic agent formulated in particles according to the present invention is enhanced by the robustness of the particles.

[0403] Ovalbumin loaded particles were produced as described in Example 1, with the flow composition adjusted as follows:

[0404] Inner aqueous core: 20 mg / mL ovalbumin (OVA) in PBS (pH 7.4, 1X)

[0405] • Solvent with polymer: 17.5% w / v PLGA (7-17 kDa, 50:50 L:G) in DMC

[0406] Aqueous extraction phase: 3% w / v PVA (9-10 kDA, 80% hydrolyzed) in DPBS

[0407] The particles were solidified as described in Example 1. The amount of encapsulated OVA was measured by disrupting a known amount of particles using a homogenizer and measuring the protein content by microBCA (ThermoFisher).

[0408] In vivo experiments were performed in 4 groups of 10 female Balb / c mice each, and each mouse received a dose of 30 μg OVA in different regimens;

[0409] ·Group 1( Fig.18 , green in the original color image):

[0410] The mice were given an initial OVA dose alone, and then a booster OVA dose was given 23 weeks later.

[0411] ·Group 2 ( Fig.18 , purple in the original color image):

[0412] The priming OVA dose was encapsulated in particles as described above and ruptured prior to injection.

[0413] ·Group 3( Fig.18 , blue in the original color image):

[0414] The priming OVA dose was encapsulated in intact particles produced as described above.

[0415] · Group 4 ( Fig.18 , red in the original color image):

[0416] The priming OVA dose was mixed with empty PLGA capsules.

[0417] Blood was collected from each mouse weekly and OVA-specific IgG antibody titers were determined by ELISA. The peak antibody titers for each preparation were plotted on Fig.18In. It can be seen that due to delayed release, an enhanced humoral immune response (OVA-specific IgG antibody response) was observed for encapsulated OVA (blue). During injection, due to syringe pressure, some particles may have broken, releasing a portion of the vaccine dose, and the remaining capsules were released later. Compared with OVA alone (green), the antigen-specific antibody (Ab) titer level from complete OVA particles was 100 times higher and similar to the 2 injection regimen of OVA alone (blue compared to green). The increase in Ab titer is not due to the adjuvant effect of the particles themselves, because the OVA regimen of empty particles (red) is similar to that of OVA alone. Ruptured OVA particles (purple part) cause a higher response. Without being bound by theory, the inventors believe that this may be because PLGA fragments can be internalized by immune cells, thereby enhancing inflammation. In general, monomer immunization with encapsulated OVA provides the highest Ab titer with the least number of injections.

[0418] Example 7

[0419] This example describes the immunization of mice with a malaria vaccine by intramuscular injection of either a soluble form of R21 or R21 encapsulated within particles of the present disclosure.

[0420] To illustrate the ability of the particles to generate immune responses with different antigens, R21 was used in this example. R21 is a virus-like particle vaccine against malaria developed by the Jenner Institute at the University of Oxford.

[0421] Particles loaded with R21 were produced as described in Example 1, and the flow composition was adjusted as follows:

[0422] Internal aqueous core: 0.36 mg / mL R21 in PBS (pH 7.4, 1X)

[0423] • With polymer: 17.5% w / v PLGA (7-17 kDa MW, 50:50 L:G) in DMC

[0424] Aqueous extraction phase: 3% w / v PVA (9-10 kDA, 80% hydrolyzed) in DPBS

[0425] The particles were solidified as described in Example 1. The amount of encapsulated R21 was measured by disrupting a known amount of particles using a homogenizer and measuring the protein content by microBCA (ThermoFischer).

[0426] Eight mice were immunized intramuscularly with 1 μg of R21 in soluble form (Ag-Sol) (denoted as antigen-Ag) or with 1 μg of R21 encapsulated in the particles produced as described above (Ag-Par). Fig.19As shown, blue mice (original color images) received a single prime injection of Ag-Sol, green mice (original color images) received a two-dose prime / boost regimen of Ag-Sol, and orange mice (original color images) received a single prime injection of Ag-Sol and Ag-Par.

[0427] In the relevant groups, three weeks after the booster injection, the Ab titer of the Ag-Sol group (blue in the original color image) that only received the prime immunization decreased significantly, while the mice that received Ad-Par (orange in the original color image) maintained the same level of R21-specific Ab. This shows the advantages of the prime-boost regimen that can be achieved according to the present invention.

[0428] Ab titers were higher in the prime / boost regimen (green in the original color image); however, without being bound by theory, the inventors believe this may be due to the presence of adjuvant in the boost dose (there is no adjuvant encapsulated in the particles).

[0429] Example 8

[0430] This example demonstrates that the release characteristics of homogenous particles prepared by the present disclosure are improved relative to heterogeneous particles produced by conventional methods (batch emulsification by vortexing), with improved delayed and sharper release observed.

[0431] As described in Example 1, homogeneous particles were produced using microfluidics, and the mobile phase composition was as follows:

[0432] Inner aqueous core: 50 mg / mL dextran TRITC (40 kDa MW) in PBS (pH 7.4, 1X)

[0433] • Solvent with polymer: 17.5% w / v PLGA (7-17 kDa MW, 50:50 L:G) in DMC

[0434] Aqueous extraction phase: 3% w / v PVA (9-10 kDA, 80% hydrolyzed) in DPBS

[0435] Using the same mobile phase composition, heterogeneous particles were produced by vortexing. First, 100 μL of the aqueous phase was added to 500 μL of the solvent polymer phase and vortexed at 3000 rpm for 1 minute in a 2 mL LoBind Eppendorf. This mixture was added to 10 mL of the aqueous extraction phase in a glass vial and vortexed at 2500 rpm for 1 minute. The particles were left to stand in the large volume of the aqueous extraction phase for 24 hours to extract the solvent and harden the particles, thus producing Fig. 20A Heterogeneous population shown.

[0436] Release from each batch was assessed in vitro according to the same protocol described in Example 2 and the released fluorescent dextran-TRITC was sampled periodically.

[0437] like Fig. 20B As shown, the homogeneous particles produced by microfluidics ( Fig. 20B , red in the original color image), compared to the heterogeneous particles produced by vortexing ( Fig. 20B , blue in the original color image) batch rapidly released most of its payload within the first 10 days without any delay. Therefore, homogenous particles are more suitable for booster delivery under vaccination due to their ability to delay release.

[0438] This experiment demonstrates that while current malaria vaccination requires a multi-dose injection regimen, encapsulation using the particles disclosed herein reduces the need for multiple injections.

[0439] Example 9

[0440] The ability of R21 (see Example 7) to elicit immunogenicity after encapsulation and in vivo injection was evaluated by immunizing mice using the following protocol:

[0441] Primary immunization: single injection of R21 alone in PBS (suspension)

[0442] Particles: A single injection of R21 alone encapsulated in approximately 50,000 LCSS particles made of 7-17 kDa MW, 50:50 L:G PLGAR502, also referred to herein as short formulation

[0443] Prime+Particles: A single injection of R21 alone in PBS and a combination of R21 short formulation particles encapsulated in approximately 50,000 LCSS

[0444] Prime / boost: Two injections of R21 alone in PBS 3 weeks apart (titers were measured only at week 5, before peak antibody titers were observed)

[0445] Mice receiving either the particle or prime+particle regimen were injected with -50,000 particles to match the soluble priming dose of R21 (1 μg), with each particle containing only a fraction of the total dose.

[0446] BALB / c mice (n=8 per group) were immunized with four different regimens by intramuscular injection. Injections were performed using a 100 μL Hamilton glass syringe sterilized with ethanol and a 25G needle. The injection volume was 50 μL, with 0.5% carboxymethyl cellulose in PBS as a viscosity enhancer to prevent LCSS particles from aggregating in the syringe. Immunogenicity was monitored by weekly blood sampling, followed by quantification of IgG by indirect ELISA using a portion of the C-term fragment (a peptide library covering 67 amino acids, "Cterm") as an antigen. The peak antibody titers observed in each mouse were compared weekly, and a threshold of 0.5-fold change in antibody titer was used to determine non-inferiority between groups.

[0447] The immunogenicity of R21 is retained when encapsulated within particles

[0448] The particle and prime+particle regimens were compared with the prime and prime / boost control regimens. Fig.21 These curves do not indicate any qualitative adverse effect of the encapsulation process on the immune response. On the contrary, when comparing the prime and particle regimens, the response to the Cterm fragment appears to be enhanced by encapsulation.

[0449] Fig. 22 The maximum IgG peak observed for each preparation and the multiple changes of the average peak log titer between the soluble preparation and the capsule preparation of the dose matching are shown. Table 1 records the estimated geometric mean and confidence interval of the four schemes. By permutation ANOVA test, the Cterm-specific IgG levels induced by different schemes are significantly different (NANP p=0.0016, Cterm p=0.0002). Compared with the corresponding primary immunization control alone, the encapsulation of R21 makes the Cterm-specific IgG titer 11 times higher (95%CI 4.8-25, p<0.05). In contrast, there is not enough evidence to show that the primary immunization + particle scheme is not inferior to the primary immunization / boosting scheme, and the multiple change of the Cterm reaction is 1 (95%C10.3-3.3, p>0.05).

[0450] Table 1: Geometric mean estimates of peak antibody titers for the unadjuvanted R21 regimen

[0451]

[0452] The adjuvant effect of the adjuvant is maintained when co-encapsulated within the particle

[0453] Protein antigens require the addition of adjuvants to increase their immunogenicity. In this study, adjuvant, a liposome-based adjuvant containing the saponin QS21 and a TLR-4 agonist integrated into the liposome bilayer, was used. To evaluate the retention of its adjuvant effect when co-encapsulated with R21 in LCSS particles, the immune responses from the above prime and particle regimens were compared with those of a similar regimen that included an adjuvant. They were designated as Prime-Adj and Particle-Adj, respectively. For the Particle-Adj regimen, the same short formulation as for the Particle regimen was used.

[0454] Mice receiving the Particle-Adj regimen were injected with ~100,000 LCSS particles to match the soluble priming dose of R21 in the Prime-Adj regimen (2 μg total). BALB / c mice were immunized with different regimens (Prime-Adj n=16, otherwise n=8) by intramuscular injection. Injections were performed using a 100 μL Hamilton glass syringe sterilized with ethanol and a 25G needle. The injection volume was 50 μL, which contained 0.5% carboxymethylcellulose in PBS as a thickener to prevent particles from aggregating in the syringe. Since the adjuvant is provided at a fixed concentration, and in order to avoid injecting too many particles and causing needle clogging, the amount of adjuvant co-encapsulated with R21 is lower than the amount provided in the unencapsulated regimen. Therefore, mice in the Particle-Adj regimen received 1 / 7 of the standard adjuvant dose. The resulting immune response kinetic curve is shown in Fig.23 middle.

[0455] The addition of adjuvant increased the immune response against both epitopes in the prime regimen starting from day 7 after immunization. Co-encapsulation of adjuvant and R21 in LCSS particles delayed the immune response against both epitopes compared with the prime-Adj regimen.

[0456] Fig.24 The maximum IgG peak observed for each formulation and the fold change in mean peak log titer between dose-matched soluble and capsule formulations are shown. Table 2 records the estimated geometric means and confidence intervals for the different regimens. By permutational ANOVA test, the addition of adjuvant induced significant differences in Cterm-specific IgG levels between encapsulated and non-encapsulated formulations (NANP p < 2 × 1 μ -16 , Cterm p<2×10 -16 Encapsulation significantly affected the Cterm-specific responses of both adjuvanted and non-adjuvanted formulations (p < 2 × 10 -16 The interaction between the effect of encapsulation and the addition of adjuvant was not significant. Encapsulation of R21 with adjuvant increased immunogenicity compared to the unencapsulated control, with an 11-fold change in Cterm-specific IgG titers (95% CI 7-17, p<0.05).

[0457] Table 2: Geometric mean estimates of peak antibody titers for the R21 adjuvant regimen

[0458]

[0459] In subsequent experiments, adjuvant was used at 1 / 7 of the full dose when co-encapsulated in the particle regimen.

[0460] Different particle formulations affect immunogenicity and delayed immune response

[0461] To investigate the effect of encapsulation on the immunogenicity of different particle formulations and the delay of immune responses, data from the prime-Adj and particle-Adj (short formulation) regimens were combined with data from two additional formulations of particles co-encapsulating R21 and split doses (1 / 7) of adjuvant. Immune responses were monitored by weekly quantification of antibody titers. A summary of the treatment regimens for each mouse and their designations in the figures is provided below:

[0462] Short: A single injection of R21 and a partial adjuvant dose encapsulated in LCSS particles made of 7-17 kDa Mw, 50:50 L:G PLGA R502 resulted in a delay of 21 days observed in vitro.

[0463] • Middle: A single injection of R21 and a partial adjuvant dose encapsulated in LCSS particles made of 54-69 kDa Mw, 50:50 L:G PLGAR505 resulted in an observed delay of 33 days in vitro.

[0464] Long: A single injection of R21 and a partial adjuvant dose encapsulated in LCSS particles made of 24-38 kDa Mw, 75:25L:G PLGA R753S resulted in a delay of 77 days observed in vitro.

[0465] • Prime: Single injection of R21 and full standard adjuvant dose in PBS.

[0466] Mice receiving the particle regimen were injected with 100,000 LCSS particles to match the soluble priming dose of R21 (here 2 μg). BALB / c mice were immunized with different regimens by intramuscular injection (primary n=16, other regimens n=8). Injections were performed using a 100 μL Hamilton glass syringe and a 25G needle sterilized with ethanol. The injection volume was 50 μL, with 0.5% carboxymethylcellulose in PBS as a viscosity enhancer to prevent particles from aggregating in the syringe.

[0467] Fig.25 Longitudinal immunoreaction kinetics of NANP- and Cterm-specific responses are shown, with corresponding generalized additive models (GAM) fits performed on log-transformed data.

[0468] The peak antibody titers and the time to reach 50% peak titers estimated from the fits are summarized in Table 3 with their 95% CIs and are shown in Fig.26 The time required to reach 50% of the peak titer (represented by the vertical bars) was chosen as a proxy for delayed immune response, as a 0.5-fold change was determined as the threshold for non-inferiority.

[0469] Table 3: Peak antibody titers and estimated time to reach 50% peak titers for NANP-specific and Cterm-specific responses to different particle formulations and unencapsulated controls

[0470]

[0471] a Geometric mean. b average value.

[0472] like Fig. 27 As shown, the encapsulation effect of R21 with adjuvant was tested for non-inferiority immunogenicity (by comparing peak antibody titers of the encapsulated regimen with the prime regimen, with a non-inferiority threshold of 0.5-fold) and delay in the onset of the immune response, indicating the lag time in the booster dose release. Differences in the delay of the immune response between the different particle formulations were then estimated.

[0473] In the short formulation, encapsulation improved immunogenicity with 12-fold higher Cterm-specific peak antibody titers compared to unencapsulated controls (95% CI 6.8-23, p<0.05). Encapsulation in short particles prolonged the delay in the onset of immunogenicity, increasing the time to reach 50% peak titers by 2.3 weeks (95% CI 1.5-3, p<0.05) for Cterm-specific antibody titers compared to unencapsulated controls ( Fig. 27 ). In the medium formulation, encapsulation resulted in an increase in Cterm-specific immunogenicity, with a 4.6-fold higher Cterm-specific peak antibody titer (95% CI 1.7-13, p < 0.05). Encapsulation in medium particles prolonged the delay of immunogenicity compared to the unencapsulated control, increasing the time to 50% peak titer by 3.7 weeks for Cterm-specific antibody titers (95% CI 3.1-4.3, p < 0.05). Finally, in the long formulation, encapsulation in long particles provided a significant extension of the delay of immunogenicity compared to the unencapsulated control, increasing the time to 50% peak titer by 13 weeks for Cterm-specific antibody titers (95% CI 13-14, p < 0.05).

[0474] The particle formulations induced significantly different delays in Cterm-specific immune responses from each other. Thus, the medium particle formulation made from higher Mw PLGA extended the time to reach 50% peak titer by 1.4 weeks (95% CI 0.5-2.3, p<0.05) compared to the short particle formulation. Similarly, the long particle formulation made with higher L:G ratio PLGA extended the time to reach 50% peak titer of Cterm-specific response by 9.5 weeks (95% CI 8.7-10, p<0.05) compared to the medium particle formulation.

[0475] Prime + particle formulation provides superior immunogenicity over prime alone

[0476] The combination of the R21 vaccine and adjuvant showed high immunogenicity and protection in malaria challenge studies when administered in a multiple injection regimen. To evaluate the potential benefits of a combined injection of priming and encapsulated doses (referred to as the "priming + PAR" regimen) relative to a single matched dose injection (referred to as the "priming" regimen), short and medium particle formulations were evaluated in parallel studies. For each study, BALB / c mice were injected with 3 different methods and immunogenicity and efficacy were compared in a malaria challenge model. The regimen is as follows:

[0477] • Prime: Single injection of R21 and adjuvant in PBS, matching the total amount received in the Prime+PAR regimen (2 full doses of R21, 1+1 / 7 dose of adjuvant).

[0478] Prime / boost: Prime with R21 and adjuvant in PBS, boost with R21 and adjuvant in PBS. For short-term studies, boost is given two weeks after the prime; for medium-term studies, boost is given three weeks after the prime.

[0479] • Prime + short PAR / prime + medium PAR: single injection of R21 and adjuvant in PBS, and R21 was co-encapsulated with a partial dose (1 / 7) of adjuvant in short-formulation or medium-formulation LCSS particles.

[0480] Blood samples were collected weekly to track immune responses until the malaria challenge phase. Fig.28 shown.

[0481] like Fig.29 As shown, all three regimens in the short-term study had similar Cterm immune responses in the first two weeks. As expected, the booster dose produced a rise in antibody titers in the prime / boost regimen, which was similar to the Cterm-specific antibody titers in the prime+short PAR regimen. The Cterm-specific response in the prime regimen reached a plateau after week 3. Fig.30The maximum Ig peak observed for each regimen is shown, as well as the multiple changes in the average peak log titer between the encapsulated regimen and the unencapsulated control. Table 4 records the estimated geometric mean and confidence interval of the three regimens. Compared with the primary immunization regimen, the primary immunization + short PAR regimen brings higher Cterm-specific immunogenicity, with a 20-fold higher antibody titer (95% CI, 12-28, p < 0.05); compared with the primary immunization / boosting regimen, the primary immunization + short PAR regimen brings higher Cterm-specific immunogenicity, with a 2.7-fold higher antibody titer (95% CI, 1.7-4.3, p < 0.05).

[0482] Compared with the prime regimen, the prime + mid-PAR regimen produced a stronger immune response to the Cterm epitope ( Fig.31 ). However, after week 2, the kinetics diverged, with titers in the prime+medium PAR group continuing to rise, while titers in the prime regimen reached a plateau. The prime+medium PAR regimen ultimately reached a plateau between the prime and prime / boost groups. Fig.32 The maximum IgG peak observed for each regimen is shown, as well as the multiple changes in the average peak log titer between the encapsulated regimen and the unencapsulated control. Table 4 records the estimated geometric mean and confidence interval of the three regimens. Compared with the primary immunization alone, the primary immunization + medium PAR induced a higher immunogenicity against the Cterm epitope, with 2.6 times higher NANP-specific antibodies (95% CI, 1.6-4.2, p < 0.05), and 15 times higher Cterm-specific antibody titers (95% CI, 11-21, p < 0.05). In contrast, compared with the primary immunization / boosting regimen, Cterm-specific immunogenicity was lower, with antibody titers changing 0.4 times (95% CI, 0.3-0.6, p < 0.05).

[0483] The estimated maximum observed peak titers for the short-term and mid-term studies are summarized in Table 4 below.

[0484] Table 4: Geometric mean estimates of peak antibody titers in short and medium formulation studies

[0485]

[0486] Encapsulation improves efficacy of single-dose vaccine

[0487] For each study, animals in the prime and prime+PAR regimens were divided into two groups of 8 mice each for challenge at two time points: at weeks 3 and 4 post-immunization in the short-term study and at weeks 5 and 6 post-immunization in the mid-term study, with the challenge period shared between the studies. This corresponds to the estimated time interval covering their respective peak antibody titers after particle release. Prime / boost controls (n=8 per study) were challenged only once, within 2 weeks of boost. Uninfected mice were shared between the two studies (n=8 per round of challenge). The challenge design is summarized in Fig.28 middle.

[0488] Malaria challenge was performed by intravenous injection of 1000 P. falciparum sporozoites. The development of parasitemia was monitored from day 5 by assessing the presence and percentage of infected erythrocytes in peripheral blood using thin blood smears. Animals with parasitemia reaching 1% were removed. Animals that were still not infected on day 12 were considered sterile. The effect of the regimen on the time of infection was analyzed by Cox regression, and the hazard ratio was calculated and adjusted according to the attack period. The hazard ratio of the regimen of interest represents the relative risk of instantaneous infection at any given time relative to the primary immunization regimen.

[0489] For the mice in this study, a single injection of prime + short PAR provided 25% protection (4 / 16 mice), while in the prime-only group, 12.5% ​​(2 / 16 mice) survived. Prime + short PAR was more effective in delaying infection compared with prime alone ( Fig.33 ), with a risk ratio of 0.4 (95% CI 0.2-0.9, p=0.02). The prime / boost regimen provided 87.5% protection (7 / 8 mice), and the prime alone was also more effective, with a risk ratio of 0.08 (95% CI 0.01-0.6, p=0.01).

[0490] In this study, it was observed that a single injection of the prime + mid-PAR formulation resulted in 43.8% sterile protection against malaria (7 / 16 mice), compared to 13.3% sterile protection when the prime alone was injected (2 / 15 mice). The prime + mid-PAR formulation was also more effective in delaying infection compared to the prime alone ( Fig.34 ), with a hazard ratio of 0.4 (95% CI 0.2-0.9, p=0.026). The prime / boost regimen resulted in 100% sterility protection (8 / 8 mice). In the absence of infection during the prime / boost regimen, the hazard ratio could not be estimated.

[0491] discuss

[0492] R21 retains immunogenicity after encapsulation

[0493] The short-delayed release formulation LCSS particles of the encapsulated R21 vaccine produced non-inferior Cterm responses compared to the same dose of unencapsulated vaccine. This suggests that the microfluidic emulsification process and in vivo exposure to physiological conditions after injection of encapsulated R21 do not affect the immunogenicity of either epitope studied, demonstrating the utility of this technology in malaria vaccines. In addition, responses to the Cterm epitope were significantly higher in the particle regimen compared to the prime, suggesting that the kinetics of release and the delay caused by exposure to the booster vaccine dose increase the magnitude of the Cterm-specific immune response. The low immunogenicity of unadjuvanted R21 precludes comparison of the delay in generating immunogenicity and the associated peak titer differences, as all previous preclinical work with R21 included the use of adjuvants.

[0494] Co-encapsulation of R21 and a fractional dose of adjuvant in short-delayed release formulation particles demonstrated the same ability to maintain or improve immunogenicity compared to unencapsulated adjuvanted vaccine. Despite a 7-fold reduction in adjuvant dose, Cterm-specific responses were significantly increased by more than 12-fold.

[0495] The delay of Cterm-specific reactions can be modulated by LCSS particle formulation

[0496] Overall, the immunogenicity results showed that Cterm-specific responses increased with repeated exposure to the R21 vaccine, as previously reported. In fact, mice that received only a single priming injection had the lowest Cterm-specific antibody titers, while mice that also received a booster showed a significant increase in Cterm-specific immunogenicity ( Fig. 22 ). In the case of LCSS particles, the delivery kinetics of the encapsulated vaccine payload is the result of the cumulative release of each single particle, as observed in vitro. The individual release of each particle occurs after a slightly different lag, resulting in an overall release of all particles, which can last from days to weeks, depending on the LCSS particle formulation. Therefore, multiple releases of different portions of the total injected particles will allow multiple exposures to the vaccine in vivo, resulting in higher levels of Cterm-specific antibody responses than a single exposure at discrete times (such as in a priming regimen alone). Therefore, Cterm immunogenicity monitoring can be used to detect delays in vaccine release, with the time of peak antibody titers indicating the end of exposure to the vaccine. Short, medium, and long formulations of LCSS particles demonstrated the ability to induce significant delays in Cterm-specific peak responses compared to a priming regimen alone, ranging from an additional 2 weeks for the short formulation to 13 weeks for the long formulation. This delay is consistent with the order of release observed in vitro (short delay < medium delay < long delay) and is consistent with the expected release order of the PLGA composition of each formulation (increasing Mw for the medium formulation and increasing L:G for the long formulation), demonstrating the tunability of this technology.

[0497] Adding encapsulated boosters to priming regimens increases Cterm-specific immune responses

[0498] Different immunogenicity patterns were observed when the standard prime vaccine dose was co-injected with either the short- or medium-granule formulation of the booster vaccine containing a fraction (1 / 7) of the adjuvant dose. The prime + medium PAR regimen induced 15-fold higher Cterm-specific antibody titers compared with the matched-dose prime regimen alone ( Fig.32 ). Compared with the priming regimen alone, the priming + short PAR regimen induced 20-fold higher Cterm-specific antibody titers ( Fig.30 ).

[0499] Increased protection against malaria by adding an encapsulated booster to a soluble prime

[0500] Despite their different immunogenicities, the two prime + particle (short and medium) regimens provided significantly reduced risk ratios in malaria challenge experiments compared to the prime only regimen, indicating that when administered as a single injection, booster dose encapsulation enhanced the protection against malaria in mice by more than 2.5 times. It was observed that the sterile protection of the co-injected prime and particle regimen was higher than that of the prime alone, highlighting the additional efficacy provided by a single injection of particles. When the medium formulation particles were used in combination with the prime dose, 44% of the mice survived, which was twice that of the short formulation particles (25%) and 3.3 times that of the prime alone (13%).

[0501] These results demonstrate for the first time the benefits of an injectable delayed-delivery system for clinically relevant vaccines in a disease challenge model in the context of single-dose vaccine development.

[0502] definition

[0503] It is to be understood that the "embodiments" of the present disclosure may be specifically combined unless the context indicates otherwise. All specific combinations of disclosed embodiments (unless the context indicates otherwise) are further disclosed embodiments of the claimed invention.

[0504] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0505] When the term "comprising" is used in the present specification and claims, other elements or steps are not excluded. In addition, the terms first, second, third, etc. used in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable where appropriate, and that the embodiments of the invention described herein are capable of operating in other sequences than those described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the present invention. Unless expressly defined herein, all terms used herein have the same meaning as those of ordinary skill in the art of the present invention. For definitions and terminology in the field related to biological substances (such as polynucleotides and polypeptides), practitioners may pay particular attention to Sambrook et al, Molecular Cloning: A Laboratory Manual, 4 th ed., Cold Spring Harbor Press, Plainsview, New York (2012) and Ausubel et al., Current Protocols in Molecular Biology (vupplement 114), John Wiley & Sons, New York (2016). For definitions and terminology related to chemical substances, including synthetic reactions used for their synthesis, practitioners may pay special attention to "March's Advanced Organic Chemistry", Wiley, 2020.

[0506] For further information on the techniques and protocols mentioned herein, practitioners in the art can also be directed to Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0507] As used herein, "about" when referring to a measurable value, such as an amount, a duration, etc., is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% of the defined value, as such variations are suitable for performing the disclosed methods.

[0508] As used herein, "nucleotide sequence", "DNA sequence" or "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Nucleic acids can be synthesized in vitro or isolated from natural sources. Nucleic acids can also include modified DNA or RNA, such as methylated DNA or RNA, or RNA that has been post-translationally modified, such as 5'-capping with 7-methylguanosine, 3'-processing (such as cleavage and polyadenylation), and splicing. Nucleic acids can also include synthetic nucleic acids (XNA), such as hexitol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), threose nucleic acids (TNA), glycerol nucleic acids (GNA), locked nucleic acids (LNA) and peptide nucleic acids (PNA).

[0509] In the context of the present disclosure, the term "amino acid" is used in its broadest sense and is intended to include organic compounds containing amino (NH2) and carboxyl (COOH) functional groups and side chains (e.g., R groups) specific to each amino acid. In some embodiments, amino acids refer to naturally occurring Lα-amino acids or residues. Common single-letter and three-letter abbreviations of naturally occurring amino acids are used herein: A = Ala; C = Cys; D = Asp; E = Glu; F = Phe; G = Gly; H = His; I = Ile; K = Lys; L = Leu; M = Met; N = Asn; P = Pro; Q = Gln; R = Arg; S = Ser; T = Thr; V = Val; W = Trp; and Y = Tyr (Lehninger, AL, (1975) Biochemistry, 2d ed., pp. 71-92, Worth Publishers, New York). The general term "amino acid" also includes D-amino acids, retro-inversoamino acids, and chemically modified amino acids, such as amino acid analogs, natural amino acids that are not usually incorporated into proteins (such as norleucine), and chemical compounds with amino acid characteristics known in the art (such as β-amino acids). For example, analogs or mimetics of phenylalanine or proline allow the same conformational constraints of peptide compounds as natural Phe or Pro, and are included in the definition of amino acids. Such analogs and mimetics are referred to herein as "functional equivalents" of the corresponding amino acids. Other examples of amino acids are listed in Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., NY 1983, which is incorporated herein by reference.

[0510] The terms "polypeptide" and "peptide" are used interchangeably herein to refer to polymers of amino acid residues and variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers (wherein one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of corresponding naturally occurring amino acids), and also to naturally occurring amino acid polymers. Peptides can be made using recombinant techniques, for example, by expression of recombinant or synthetic polynucleotides. Recombinantly produced peptides are typically substantially free of culture medium, for example, culture medium is less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0511] The term "protein" is used to describe a folded polypeptide having a secondary or tertiary structure. A protein may consist of a single polypeptide or may include multiple polypeptides assembled into a polymer. A polymer may be a homo-oligomer or a hetero-oligomer. A protein may be a naturally occurring or wild-type protein or a modified or non-naturally occurring protein. A protein may differ from a wild-type protein, for example, by the addition, substitution or deletion of one or more amino acids.

[0512] The term "alkyl" refers to a radical of the formula (CH n ) 2n+1 Examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0513] The term "aryl" refers to a substituted or unsubstituted, monocyclic or fused polycyclic aromatic group typically containing 6 to 10 ring carbon atoms. Examples include monocyclic groups such as phenyl; and fused bicyclic groups such as naphthyl and indenyl. Phenyl (benzene) is generally preferred.

[0514] As used herein, a pharmaceutically acceptable salt is a salt formed with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include: inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid; and organic acids such as oxalic acid, citric acid, fumaric acid, maleic acid, malic acid, ascorbic acid, succinic acid, tartaric acid, benzoic acid, acetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid or p-toluenesulfonic acid. Pharmaceutically acceptable bases include alkali metal (e.g., sodium or potassium) and alkaline earth metal (e.g., calcium or magnesium) hydroxides and organic bases (e.g., alkylamines, arylalkylamines and heterocyclic amines). Preferred hydrochlorides and acetates, particularly hydrochlorides.

[0515] Unless the context requires otherwise, the stereochemistry of the compounds disclosed herein is not limited. In particular, the particles disclosed herein may include components containing one or more chiral centers. These components can be used in enantiomerically or diastereomerically pure forms, or in the form of isomer mixtures. In addition, for the avoidance of doubt, these components can be used in the form of any tautomers. Typically, the active agents or substances described herein are at least 50%, preferably at least 60%, 75%, 90% or 95% enantiomerically or diastereomerically pure. Typically, the compounds disclosed herein include at least 60%, such as at least 75%, 90% or 95% single enantiomers or diastereomers by weight. Preferably, the disclosed compounds are substantially optically pure.

Claims

1. A method of preparing particles comprising an aqueous core encapsulated by a polymer shell, the method using a flow chip having channels including a main channel formed in the flow chip, the method comprising: Allowing a solvent phase to flow through the transmission section of the main channel, wherein the solvent phase carries droplets of an aqueous phase, wherein the aqueous phase comprises a pharmaceutical agent and an aqueous solvent, and the solvent phase comprises a polymer and a non-aqueous solvent; allowing an extraction phase to flow through an extraction phase inlet channel in the flow chip, the extraction phase comprising an extraction solvent, wherein the extraction phase inlet channel is disposed on opposite sides of the main channel, and the transport section and the extraction phase inlet channel lead to an extraction phase junction section of the main channel, thereby forming solvent phase droplets encapsulating the aqueous phase droplets in the extraction phase; and Allowing the extraction phase entrained with the solvent phase droplets to flow through an extraction section of the main channel extending from the extraction phase intersection section; and extracting the non-aqueous solvent of the aqueous phase so as to form particles having an aqueous core formed by droplets of the aqueous phase and a shell formed by the polymer; The main channel increases in height at or downstream of the location where the transmission section leads to the extraction phase intersection section.

2. The method according to claim 1, wherein: The main channel increases in height where the transfer section leads to the extraction phase junction section.

3. The method according to claim 1, wherein: The main channel increases in height downstream of where the transfer section opens into the extraction phase junction section.

4. A method according to any one of the preceding claims, wherein: The main channel has a height in the range of 5 μm to 250 μm before the height increase.

5. A method according to any one of the preceding claims, wherein: The main channel has a height in the range of 20 μm to 500 μm after the height increase.

6. A method according to any one of the preceding claims, wherein: The height of the main channel increases by at least 15 μm.

7. A method according to any one of the preceding claims, wherein: The extraction phase inlet channel has a width in the range of 20 μm to 500 μm.

8. A method according to any one of the preceding claims, wherein: The extraction section includes an extraction nozzle section downstream of the extraction phase intersection section, and a width of the extraction nozzle section increases with a distance from the extraction phase intersection section.

9. The method according to claim 8, wherein: The extraction nozzle section includes a neck section and an expansion section downstream of the neck section, and the width of the extraction nozzle section increases in the expansion section.

10. The method according to claim 9, wherein: The neck section of the extraction nozzle section has a width in the range of 10 μm to 200 μm; The neck section of the extraction nozzle section has a length ranging from 10 μm to 200 μm; The expansion section of the extraction nozzle section has a maximum width in the range of 100 μm to 1000 μm; and / or The expanded section of the extraction nozzle section has a length in the range of 10 μm to 1000 μm.

11. A method according to any one of the preceding claims, wherein: The flow rate of the extraction phase through the extraction phase inlet channel is greater than the flow rate of the solvent phase through the transfer section.

12. A method according to any one of the preceding claims, wherein: The flow rate of the solvent phase through the transfer section is in the range of 2.5 nL / s to 150 μL / s.

13. A method according to any one of the preceding claims, wherein: The flow rate of the extraction phase through the extraction phase inlet channel is in the range of 12 nL / s to 3 mL / s.

14. The method according to any one of the preceding claims, further comprising: allowing the aqueous phase to flow through the aqueous phase inlet section of the main channel; and The solvent phase is allowed to flow through a solvent phase inlet channel in the flow chip, the solvent phase inlet channel being arranged on the opposite side of the main channel, and the aqueous phase inlet section and the solvent phase inlet section leading to the solvent phase intersection section of the main channel, so that the aqueous phase droplets are formed in the solvent phase, The transfer section extends from the solvent phase intersection section.

15. The method according to claim 14, wherein: The transport section includes a solvent nozzle section downstream of the solvent phase intersection section, and a width of the solvent nozzle section increases with a distance from the solvent phase intersection section.

16. The method according to claim 15, wherein: The solvent nozzle section includes a neck section and an expansion section downstream of the neck section, and the increase in width occurs in the expansion section.

17. The method of claim 16, wherein: The neck section of the solvent nozzle section has a width in the range of 5 μm to 200 μm; The neck section of the solvent nozzle section has a length in the range of 10 μm to 200 μm; The expanded section of the solvent nozzle section has a maximum width in the range of 100 μm to 500 μm; and / or The expanded section of the solvent nozzle section has a length in the range of 10 μm to 500 μm.

18. The method according to any one of claims 14 to 17, wherein: The flow rate of the solvent phase through the solvent phase inlet channel is greater than the flow rate of the aqueous phase through the aqueous phase inlet section.

19. The method according to any one of claims 14 to 18, wherein: The flow rate of the aqueous phase through the aqueous phase inlet section is in the range of 0.25 nL / s to 30 μL / s.

20. A method according to any one of the preceding claims, wherein: The surface of the main channel in the flow chip upstream of the extraction phase junction is hydrophobic.

21. A method according to any one of the preceding claims, wherein The extraction solvent is hydrophilic, The surface of the extraction phase inlet channel is hydrophilic, and The surface of the main channel of the flow chip at and downstream of the extraction phase junction is hydrophilic.

22. A method according to any one of the preceding claims, wherein: The channel has a planar extent.

23. A method according to any one of the preceding claims, wherein: The flow chip is formed by multi-layer molding.

24. A method according to any one of the preceding claims, wherein: The flow chip includes a main body, which includes PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), polycarbonate (PC), cyclic olefin copolymer (COC) or glass, or consists of PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), polycarbonate (PC), cyclic olefin copolymer (COC) or glass.

25. A method according to any one of the preceding claims, wherein: The agent is selected from the group consisting of: immunogenic agents, analgesics, antibiotics, antithrombotic agents, antidepressants, anticancer agents, antiepileptic agents, anti-inflammatory agents, antipsychotic agents, antiviral agents, sedatives, steroids, antidiabetic agents, cardiovascular agents, and drugs for pain control, treatment of skin disorders, and treatment of brain diseases; Preferably, the pharmaceutical agent is a vaccine agent.

26. A method according to any one of the preceding claims, wherein: The aqueous phase solvent comprises an aqueous solution having a pH of about 4 to about pH 10; Optionally, wherein the aqueous solvent comprises one or more buffer salts and / or one or more gelling agents and / or one or more stabilizers.

27. A method according to any one of the preceding claims, wherein: The polymer comprises or consists of one or more biodegradable polymers, wherein the one or more biodegradable polymers are selected from aliphatic polyesters, aromatic copolyesters, polyurethanes, polycarbonates, polyamides, poly(ester-amides), polyanhydrides, polysaccharides, and blends thereof or copolymers thereof, and Preferably, the one or more biodegradable polymers are selected from poly(lactic acid-co-glycolic acid) (PLGA), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly(butylene succinate), poly(dioxanone) (PPDO), poly(hydroxybutyrate) (PHB), poly(butylene adipate-co-butylene terephthalate) (PBAT), chitosan, cellulose, hyaluronic acid, and blends thereof or copolymers thereof.

28. A method according to any one of the preceding claims, wherein: The non-aqueous solvent is selected from dimethyl carbonate (DMC), dichloromethane (DCM), toluene, chloroform, n-hexane, ethyl ether, benzene, n-butanol, butyl acetate, carbon tetrachloride, cyclohexane, 1,2-dichloroethane, ethyl acetate, heptane, methyl tert-butyl ether, methyl ethyl ketone, pentane and dichloroethylene, and mixtures thereof.

29. A method according to any one of the preceding claims, wherein: The extraction solvent comprises an aqueous solution of about pH 4 to about pH 10; Optionally, wherein the extraction solvent comprises one or more buffer salts, one or more surfactants, one or more viscosity enhancing agents and / or one or more osmotic pressure regulating agents.

30. A method according to any one of the preceding claims, wherein: The non-aqueous solvent is extracted by evaporation or by liquid phase extraction into the aqueous solvent.

31. A method according to any one of the preceding claims, wherein: The aqueous solvent and the non-aqueous solvent are immiscible.

32. A flow chip for preparing particles, the particles comprising an aqueous core encapsulated by a polymer shell, the flow chip having channels formed in the flow chip, the channels comprising a main channel and an extraction phase inlet channel disposed on opposite sides of the main channel, The main channel comprises: Transmission segment, an extraction phase intersection section, wherein the transmission section and the extraction phase inlet passage lead to the extraction phase intersection section; and Extraction segments extending from the extraction intersection segment, The main channel increases in height at or downstream of the location where the transmission section leads to the extraction phase intersection section.

33. The device according to claim 32, wherein The channel further comprises a solvent phase inlet channel on an opposite side of the main channel, and The main channel further comprises: Aqueous phase inlet section; and a solvent phase intersection section, wherein the aqueous phase inlet section and the solvent phase inlet channel lead to the solvent phase intersection section, and a transmission section extending from the solvent phase intersection section.

34. An aqueous core-polymer shell particle comprising: an aqueous core comprising a pharmaceutical agent dissolved or dispersed in an aqueous solvent; and A homogeneous solid biodegradable polymer shell encapsulating the aqueous core.

35. The particle of claim 34, wherein: The medicament is as defined in claim 25; and / or The aqueous solvent is as defined in claim 26; and / or The polymer shell comprises or consists of one or more polymers as defined in claim 27 .

36. The particle according to claim 34 or 35, wherein The particle has a diameter of about 5 μm to about 500 μm; and / or wherein the polymer shell has a thickness of about 0.1 μm to about 100 μm; Preferably, the diameter of the particle is about 20 μm to about 150 μm; and / or the thickness of the polymer shell is about 1 μm to about 20 μm.

37. A population of particles according to any one of claims 34 to 36, wherein at least 90% of the particles in the population are characterized by comprising: The average diameter is a single spherical aqueous core volume having a range of from about 1 μm to about 300 μm, and wherein the smallest diameter of the aqueous core is at least 70% of the largest diameter of the aqueous core; and A biodegradable polymer shell having a thickness of about 0.1 μm to about 100 μm, wherein the thickness of the thinnest portion of the polymer shell is at least 70% of the thickness of the thickest portion of the polymer shell.

38. A pharmaceutical composition comprising a plurality of particles according to any one of claims 34 to 36 or a population of particles according to claim 37, and one or more pharmaceutically acceptable excipients, diluents or adjuvants.

39. The pharmaceutical composition according to claim 38, wherein The composition includes an additional pharmaceutical agent.

40. A prime / boost vaccine composition comprising: (i) a plurality of particles according to any one of claims 34 to 36 or a population of particles according to claim 37, wherein each of the particles comprises an agent as a first immunogenic agent; and (ii) a second immunogenic agent; The first immunogenic agent and the second immunogenic agent are the same or different.

41. A particle according to any one of claims 34 to 36, a population of particles according to claim 37 or a composition according to any one of claims 38 to 4, for use in medicine.

42. A composition comprising a plurality of particles according to any one of claims 34 to 36 and optionally further comprising one or more additional therapeutic agents, for use in a method of vaccination, preferably a prime / boost vaccination method.

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