Micromolded or 3D printed pulsed release vaccine formulations

Abstract

To provide an emulsion-based and micro-molded ("MM"), or three-dimensionally printed ("3DP") polymer preparation for single injection of antigen that is released preferably in two or more periods of time.SOLUTION: A preparation is preferably formed of biocompatible and biodegradable polymer. Separate regions for encapsulating antigen exist in the preparation alone or in combination with other antigen, adjuvant, stabilizer and release regulator. The antigen exists preferably in a filler at the time of administration or exists on the surface of the preparation for immediate release, then, incorporated into the preparation in order to be released in 10 to 45 days after the initial release of antigen, and for release of antigen at the interval of 10 to 90 days in one or more periods of time as needed.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention is generally within the field of injectable vaccine formulations that provide multiple releases of vaccine. [Background technology]

[0002] 2. Background of the Invention Vaccines typically include an initial dose of antigen followed by one or more booster doses at prescribed times, typically 10-60 days after the initial dose. In most of the world, the need to administer booster doses clearly limits the practical utility of vaccines as well as increasing the expense and difficulty in agricultural application.

[0003] Polymeric microspheres have the potential to be effective vaccine delivery vehicles. They have the ability to enhance targeting of antigen-presenting cells (APCs) and have the potential for controlled, sustained release of antigens, thereby potentially eliminating the need for multiple vaccinations. Furthermore, the polymer matrix can act as a shield from the hostile external environment and has the potential to reduce adverse reactions and suppress problems caused by vaccine strains in immunocompromised individuals. PLGA microspheres have been developed for single immunization with and without burst release. Given the biodegradable nature and sustained release properties offered by PLGA, microspheres formulated from PLGA may be useful for vaccine delivery. Kirby et al., Chapter 13: Formation and Characterization of polylactide-co-galactide PLGA microspheres (2013) summarize the properties of PLGA-based microparticles. PLGA-based microparticles are traditionally produced by double emulsion solvent evaporation, nanoprecipitation, cross-flow filtration, salting out technique, emulsion diffusion method, jet milling and spray drying. Kirby et al., Chapter 13: Formation and Characterization of polylactide-co-galactide PLGA microspheres (2013) summarize the properties of PLGA-based microparticles. The effectiveness of PGLA particles in the preparation of PGLA nanoparticles has been summarized in and Characterization of polylactide-co-galactide PLGA microspheres (2013). PLGA microspheres can also be formulated to incorporate various moieties, including drugs and proteins, that can act as adjuvants. It has been contemplated that the PGLA particles produced by these methods can be lyophilized and stored for later use and delivery.

[0004] Hanes et al., Adv. Drug. Del. Rev., 28:97-119 (1997) report on attempts to create polymeric microspheres to deliver subunit protein and peptide antigens in their native form in a continuous or pulsatile manner for weeks to months with reliable and reproducible kinetics, eliminating the need for booster immunization. Microspheres have potential as carriers for oral vaccine delivery due to their protective effect on encapsulated antigens and their ability to be taken up by the intestinal Peyer's patches. The efficacy of these optimal depot formulations for antigens can be enhanced by the co-delivery of vaccine adjuvants, including cytokines, entrapped within the polymer matrix or, alternatively, incorporated into the backbone of the polymer itself and released simultaneously with the antigen when the polymer degrades.

[0005] As reported by Cleland et al., J. Controlled Rel. 47(2):135-150 (1997), administration of subunit vaccines (e.g., gp120) for acquired immune deficiency syndrome (AIDS) can be facilitated by a single shot vaccine that mimics repeated immunization. Poly(lactic-co-glycolic acid) (PLGA) microspheres were fabricated that provide a pulsatile release of gp120. The microspheres were fabricated using a water-in-oil-in-water microencapsulation method, with either methylene chloride or ethyl acetate as the polymer solvent. The protein was released under physiological conditions in two distinct phases: an initial burst was released on the first day, and a second burst of protein was released several weeks or months later. The second burst of protein was dependent on the intrinsic viscosity of PLGA and the lactide / glycolide ratio (bulk erosion).

[0006] These studies demonstrate that it is possible to achieve a vaccine response using injectable microparticles. However, such products have not yet been approved for human or animal use. Effective antigen loading, uniformity of encapsulation and release, and extremely low levels of solvent that do not affect antigenicity are difficult to achieve.

[0007] It has been estimated that the development of heat-stable formulations could save approximately $200 million annually by eliminating the need for a "cold chain" for vaccine distribution. The difficulties in implementing these strategies stem from the lack of suitable cryoprotectant methods. Kirby et al., Chapter 13: Formation and Characterization of polylactide-co-galactide PLGA The stabilization of proteins in microspheres is problematic. Many types of stabilizing excipients have been investigated. As summarized in Kim and Pack, BioMEMS and Biomedical Nanotechnology. 1:19-50 (2006). In addition, the type of polymer used for microsphere fabrication, its degradation rate, the acidity of the degradation products, hydrophobicity, etc. may also affect the stability of the incorporated protein.

[0008] It is therefore an object of the present invention to provide an injectable polymeric formulation that provides two or more releases of the encapsulated antigen.

[0009] It is a further object of the present invention to provide an injectable polymeric formulation that does not damage and can stabilize the encapsulated antigen. It is yet a further object of the present invention to provide methods and materials for micromolding and three-dimensional printing of injectable polymer formulations, and the resulting formulations, that provide two or more releases of the encapsulated antigen. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Kirby et al., Chapter 13: Formation and Characterization of polylactide-co-galactide PLGA microspheres (2013) [Non-Patent Document 2] Hanes et al., Adv.Drug.Del.Rev., 28:97-119 (1997) [Non-Patent Document 3] Cleland et al., J.Controlled Rel.47(2):135-150(1997) [Non-Patent Document 4] Gregory et al., Frontiers in Cell and Infect.Microbio.3:Article 13(2013) [Non-Patent Document 5] Nandedkar, J. Biosci.34:995-1003(2009) [Non-Patent Document 6] Kim and Pack, BioMEMS and Biomedical Nanotechnology. 1:19-50 (2006) Summary of the Invention [Means for solving the problem]

[0011] We have developed emulsion-based and micromolded ("MM") or three-dimensionally printed ("3DP") polymer formulations for single injection of antigens that preferably release in two or more time periods. The formulations are preferably formed of biocompatible, biodegradable polymers. There are distinct regions within the formulation that encapsulate antigens alone or in combination with other antigens, adjuvants, stabilizers, and release modifiers. The antigen is preferably present in the excipient at the time of administration or on the surface of the formulation for immediate release, and is incorporated within the formulation for release 10-45 days after initial release of antigen, and optionally for one or more additional time periods at 10-90 day intervals. Antigens may be stabilized by the use of stabilizers such as trehalose glass. In a preferred embodiment for immunization against polio, the antigen is released at the time of administration and 2, 4, and 6 months thereafter. In a preferred embodiment, leakage between bursts of release is minimal, and release occurs in a narrow time frame.

[0012] Studies demonstrate the selection of polymers and solvent systems that provide separate release of antigens without overlap and with minimal degradation or damage to the encapsulated antigens. Preferred solvents include methylene chloride and chloroform, and preferred polymers are polylactic acid ("PLA"), polyglycolic acid ("PGA"), and copolymers thereof ("PLGA").

[0013] The formulation is designed for subcutaneous or intramuscular injection via needle or cannula, for local injection into mucous membrane areas such as intranasal cavity, or by scarification into epidermis.The preferred application is for administration of antigens that elicit effective immune responses against infectious agents such as bacteria, viruses, protozoa and parasites.However, the formulation may also be used to administer other therapeutic, prophylactic or diagnostic agents alone or in combination with antigens. In an embodiment of the present invention, for example, the following items are provided: (Item 1) A polymer formulation formed by emulsion formulation, 3D printing, or micromolding of a biocompatible polymer containing an antigen, optionally in combination with a stabilizer for said antigen, wherein said antigen is released over a defined period of time in an amount effective to elicit an immune response in vivo. (Item 2) A polymer formulation formed by emulsion formulation, 3D printing, or micromolding of a biocompatible polymer containing a therapeutic, prophylactic, nutraceutical, or diagnostic agent, optionally in combination with a stabilizer, wherein the agent is released from separate regions of the formulation at least two different times or with different release kinetics. (Item 3) 3. The formulation of item 2, wherein the agent is one or more antigens, and the antigens are released in an amount effective to elicit an immune response in vivo for a defined period of time. (Item 4) 4. The formulation of item 1 or 3, further comprising an effective amount of an antigen to elicit an immune response in vivo, present in the excipient, present on the surface of the formulation, or mixed with the formulation upon administration. (Item 5) 3. The formulation according to item 1 or 2, further comprising a stabilizing agent selected from the group consisting of sugars, oils, lipids, and carbohydrates, preferably sugars, most preferably selected from the group consisting of sucrose, trehalose, and combinations thereof, optionally in the form of a sugar glass. (Item 6) 4. The formulation of item 1 or 3, wherein the antigen elicits an immune response against an infectious agent or a tumor. (Item 7) 7. The formulation of item 6, wherein the infectious agent is a virus, a bacterium, a fungus or a protozoan. (Item 8) 8. The formulation of item 7, wherein the virus is selected from the group consisting of polio, influenza, hepatitis, rotavirus, measles, mumps, rubella, and chickenpox. (Item 9) 8. The formulation of item 7, wherein the bacteria is selected from the group consisting of Diptheria, Pertussis, Clostridium tetani, Streptococcus pneumoniae, and Neisseria meningitidis. (Item 10) 7. The formulation of item 6, wherein the antigen is a tumor antigen that selectively elicits a T cell response against a tumor. (Item 11) 3. The formulation according to item 1 or 2, wherein the polymer is biodegradable by hydrolysis, preferably a polyester, most preferably selected from the group consisting of poly(lactic acid), poly(glycolic acid), and copolymers thereof. (Item 12) 3. The formulation according to item 1 or 2, providing release at intervals of 10 to 90 days, preferably at intervals of 30 to 60 days. (Item 13) 3. The formulation according to item 1 or 2, which is in the form of a microparticle, microcapsule or microsphere. (Item 14) 3. The formulation according to item 1 or 2, wherein the formulation is for injection. (Item 15) 3. The formulation according to item 1 or 2, wherein the formulation is implantable. (Item 16) 3. The formulation according to item 1 or 2, wherein the formulation can be applied to a mucosal surface selected from the group consisting of the nose, lungs, mouth, vagina and rectum. (Item 17) 17. A method for delivering a therapeutic, prophylactic, nutraceutical or diagnostic agent to an individual in need thereof, comprising administering to said individual a formulation according to any of items 1 to 16. (Item 18) 18. The method of claim 17, wherein the formulation is a polymer formulation formed by three-dimensional printing or micromolding of a biocompatible polymer containing the antigen, optionally in combination with a stabilizer for the antigen, wherein the antigen is released in an amount effective to elicit an immune response in vivo for a defined period of time. (Item 19) 19. The method of claim 18, wherein the antigen is released at monthly intervals of two or more months, and the antigen is polio or DPT. [Brief description of the drawings]

[0014] [Figure 1] Figure 1 is a bar graph showing the effect of decreasing excipient:vaccine ratios on the percentage of D antigen retained after drying 26x trivalent IPV on PLA for 16 hours at room temperature and humidity. Excipients: 10% sorbitol, 8.5% MSG, 8.5% MgCl2. [Diagram 2] Figure 2 is a line graph showing the long-term stability of lyophilized IPV (types I, II, and III) with excipients in a humid environment at 37°C. Stability is expressed as percent recovery (% recovery) over time (days). In the line graph, the line for type I IPV is designated (1), the line for type II IPV is designated (2), and the line for type III IPV is designated (3). [Diagram 3]3A and 3B are graphs of the release over time (weeks) of a model protein, bovine serum albumin ("BSA"), from PLLA (50 kD) (FIG. 3A, μg / 10 mg microspheres; FIG. 3B, % BSA) for 5% BSA and 0.5% BSA. [Figure 4] 4A and 4B are graphs of the release over time (weeks) of a model protein, bovine serum albumin ("BSA"), from PLLA (100 kD) (FIG. 4A, μg / 10 mg microspheres; FIG. 4B, % BSA) for 5% BSA and 0.5% BSA. [Diagram 5] 5A and 5B are graphs of the release over time (weeks) of a model protein, bovine serum albumin ("BSA"), from PLLA (300 kD) (FIG. 5A, μg / 10 mg microspheres; FIG. 5B, % BSA) for 5% BSA and 0.5% BSA. [Figure 6] Figures 6A and 6B are graphs of the release over time (weeks) of the model protein bovine serum albumin ("BSA") from P(d,l)LA (20 kD) (Figure 6A, μg / 10 mg microspheres; Figure 6B, % BSA) for 5% BSA and 0.5% BSA. [Figure 7] 7A and 7B are graphs of protein release over time (weeks) for the PLGA formulation (FIG. 7A) and for the bolus injection (FIG. 7B). [Figure 8] 8A and 8B are graphs of the release over time (weeks) of 0.5% model protein bovine serum albumin ("BSA") compared to the release of 0.5% of another model protein ovalbumin ("OVA") from carboxyl-derivatized PLGA (50:50) (20 kD) (FIG. 8A, μg / 10 mg microspheres; FIG. 8B, % BSA). [Figure 9]9A and 9B are graphs of the release over time (weeks) of 5% model protein bovine serum albumin ("BSA") compared to the release of 5% of another model protein, ovalbumin ("OVA"), from carboxyl-derivatized PLGA (50:50) (31 kD) (FIG. 9A, μg / 10 mg microspheres; FIG. 9B, % BSA). [Figure 10] 10A and 10B are graphs of the release over time (weeks) of 0.5% model protein bovine serum albumin ("BSA") compared to the release of 0.5% of another model protein ovalbumin ("OVA") from carboxyl-derivatized PLGA (50:50) (31 kD) (FIG. 10A, μg / 10 mg microspheres; FIG. 10B, % BSA). [Figure 11] 11A and 11B are schematics of particles made by emulsion (FIG. 11A) and by 3D printing or micromolding (FIG. 11B), as well as protein release from emulsion particles (FIG. 11C) and 3DP particles (FIG. 11D) modeling protein release from a bolus injection. FIG. 11E is a schematic of a burst based on polymer degradation over a one month period simulating a bolus injection. [Figure 12] 12A-12D are schematic diagrams of the 3D printing process: creating the structure of PLGA particles (FIG. 12A), loading the particles with drugs or proteins (FIG. 12B), drying the drugs or proteins (FIG. 12C), and encapsulating the particles (FIG. 12D). [Figure 13] FIG. 13 is a schematic diagram of waveform parameters that should be optimized to produce uniform, single droplets of "ink" (polymer solution) from a piezoelectric nozzle jet during printing. [Diagram 14AB] 14A-14C are graphs of the percent of D antigen (IPV types I, II, and III) retained after lyophilization with the sugar excipients 1 M trehalose, 1 M sucrose, and 3 M sucrose followed by incubation at 4° C., 25° C., or 37° C. [Figure 14C]14A-14C are graphs of the percent of D antigen (IPV types I, II, and III) retained after lyophilization with the sugar excipients 1 M trehalose, 1 M sucrose, and 3 M sucrose followed by incubation at 4° C., 25° C., or 37° C. [Figure 15A] Figures 15A and 15B are graphs of the percent of D antigen (IPV types I, II, and III) retained after lyophilization with 0, 0.5, 0.75, 1, or 1.25 M trehalose, 1, and then incubation at 4°C (Figure 15A) or 25°C (Figure 15B). [Figure 15B] Figures 15A and 15B are graphs of the percent of D antigen (IPV types I, II, and III) retained after lyophilization with 0, 0.5, 0.75, 1, or 1.25 M trehalose, 1, and then incubation at 4°C (Figure 15A) or 25°C (Figure 15B). [Figure 16A] Figures 16A and 16B are graphs of the percent of D antigens (IPV types I, II, and III) retained after mixing with solvent until the solvent evaporates, using tetrafluoroethylene ("TFE"), dichloromethane ("DCM"), or TFE and DCM as the solvent, without a sugar excipient (Figure 16A) or with 0.75 M trehalose as the excipient (Figure 16B). [Figure 16B] Figures 16A and 16B are graphs of the percent of D antigens (IPV types I, II, and III) retained after mixing with solvent until the solvent evaporates, using tetrafluoroethylene ("TFE"), dichloromethane ("DCM"), or TFE and DCM as the solvent, without a sugar excipient (Figure 16A) or with 0.75 M trehalose as the excipient (Figure 16B). [Figure 17A]Figure 17A is a graph of the percent of D antigens (types I, II, and III IPVs) retained after printing IVPs with 0M sugar, 0.5M trehalose, or 0.5M trehalose·sucrose onto 3D-printed PLA substrates and drying at 25°C and 22% relative humidity. Figure 17B is a graph of the percent of D antigens (types I, II, and III IPVs) retained after printing IVPs with 0.25M trehalose or 0.5M trehalose·sucrose onto 3D-printed PLA substrates and drying at 25°C and 10.7% relative humidity. [Figure 17B] Figure 17A is a graph of the percent of D antigens (types I, II, and III IPVs) retained after printing IVPs with 0M sugar, 0.5M trehalose, or 0.5M trehalose·sucrose onto 3D-printed PLA substrates and drying at 25°C and 22% relative humidity. Figure 17B is a graph of the percent of D antigens (types I, II, and III IPVs) retained after printing IVPs with 0.25M trehalose or 0.5M trehalose·sucrose onto 3D-printed PLA substrates and drying at 25°C and 10.7% relative humidity. [Figure 18A] Figures 18A and 18B are graphs of the percent of D antigen retained after lyophilization with 0.5 or 0.75 M trehalose followed by incubation at 25°C (Figure 18A), or after lyophilization with pipetted or jetted 0.5 M trehalose·sucrose followed by drying at 25°C and 10.7% relative humidity (Figure 18B). [Figure 18B] Figures 18A and 18B are graphs of the percent of D antigen retained after lyophilization with 0.5 or 0.75 M trehalose followed by incubation at 25°C (Figure 18A), or after lyophilization with pipetted or jetted 0.5 M trehalose·sucrose followed by drying at 25°C and 10.7% relative humidity (Figure 18B). [Fig. 19AB]19A-19C are graphs showing anti-BSA IgG (antibody) titers (in log2 values) plotted for the groups presented in Table 3 at 1 week (FIG. 19A), 2 weeks (FIG. 19B), and 4 weeks (FIG. 19C) after injection. The negative controls are all zero, and the microsphere formulations produce equivalent or stronger responses compared to the bolus control. (F4 and F8 are empty microspheres, containing no drug; see Table 4). [Figure 19C] 19A-19C are graphs showing anti-BSA IgG (antibody) titers (in log2 values) plotted for the groups presented in Table 3 at 1 week (FIG. 19A), 2 weeks (FIG. 19B), and 4 weeks (FIG. 19C) after injection. The negative controls are all zero, and the microsphere formulations produce equivalent or stronger responses compared to the bolus control. (F4 and F8 are empty microspheres, containing no drug; see Table 4). [Figure 20] Figures 20A-20C are histograms showing the size distribution of microspheres prepared using formulation C (Figure 20A), formulation G (Figure 20B), and formulation E (Figure 20C). Figures 20D-20F are histograms showing the volume distribution of formulation C (Figure 20D), formulation G (Figure 20E), and formulation E (Figure 20F). [Figure 21] Figures 21A-21D are line graphs showing weekly BSA release (in percentage (%)) from microspheres formulated with 50:50 PLGA of 7-17 kDa (Figure 21A), 50:50 PLGA of 24-38 kDa (Figure 21B), 50:50 PLGA of 38-54 kDa (Figure 21C), and 75:25 PLGA of 4-15 kDa (Figure 21D) containing 0.5, 3, or 5 wt% BSA. Error bars represent standard deviation. [Figure 22]Figures 22A and 22B are line graphs showing IgG antibody titers in mouse serum against BSA released from microparticles and are plotted as geometric mean over time on a log2 scale. Figure 22A shows low dose formulations C and G compared to a series of 3 dose-matched bolus BSA injections. Figure 22B shows high dose formulation E compared to its dose-matched bolus control. Bolus BSA was injected at weeks 0, 4, and 8 in the control group. Filled symbols indicate significant differences between the groups in the same shape and unfilled symbols and the dose-matched control at that time point as determined using ANOVA analysis for A (group 3) and Student's t-test for B (group 2). One, two, and three filled symbols indicate p<0.05, p<0.01, and p<0.001, respectively, and error bars represent standard deviation. [Diagram 23] Figures 23A and 23B are dot plots showing peak titers induced by immunization with low (Figure 23A) and high (Figure 23B) doses of BSA. Peak titers for all microparticle formulations occurred at week 4, but both bolus injection groups peaked at week 10. NS represents no significant difference at the 0.05 significance level using a Student's t-test with Holm-Bonferroni correction for multiple comparisons. Adjusted p-values ​​for formulations C, G, and E are 0.0645, 0.0543, and 0.0784, respectively. [Figure 24] 24A-24C are schematic diagrams of the micromolding process showing steps in the microfabrication of the shell (FIG. 24A), filling the shell with drug (FIG. 24B), and sealing the shell with a cap (FIG. 24C). [Diagram 25] FIG. 25 is a bar graph showing the stability (% recovery) of IPV with and without gelatin, maltodextrin, pullulan, myristic acid, and Tween 80 as excipients during organic / aqueous mixing by vortexing or sonication. [Figure 26]FIG. 26 is a bar graph showing the stability (% recovery) of IPV with or without gelatin, maltodextrin, pullulan, myristic acid, and Tween 80 as excipients during organic / aqueous mixing with PLGA by vortexing or sonication (son = sonication). [Figure 27] FIG. 27 is a bar graph showing the stability (% recovery) of IPV with pullulan, pullulan / BSA, sorbitol / MSG / MgCl2 as excipients after drying by lyophilization overnight or after using the Genevac for 1 hour at 30-35° C. [Figure 28] 28 is a line graph showing the change in pH of the release medium over time (days) by PLGA particles in the presence of Al(OH)3 (line (2)), myristic acid (line (3)), and Mg(OH)2 (line (4)) as buffering agents. The line representing the change in pH of the release medium over time in the absence of buffering agents is labeled (1). [Figure 29] 29 is a line graph showing the change in pH of the release medium over time (days) by PLGA particles. The line for PLGA502H tested after buffer changes every 2-3 days is designated (1), the line for PLGA502H tested after buffer changes every 7 days is designated (2), the line for PLGA503H tested after buffer changes every 2-3 days is designated (3), and the line for PLGA503H tested after buffer changes every 7 days is designated (4). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description of the Invention I. Definition "Additive manufacturing" or "3D printing" as used herein refers to the process of creating three-dimensional solid objects of virtually any shape from a digital model. 3D printing is accomplished using an additive process, where successive layers of material are laid down in different shapes or thicknesses. In some embodiments, "3D printing" includes extrusion or solvent-based polymer-containing inks (e.g., PLGA, PLLA, etc.) that are jetted or extruded through a nozzle and solidified into the desired shape. Shape can be controlled in the x, y, and z directions.

[0016] As used herein, "micromolding" generally refers to a process suitable for producing parts or devices on a microscale, or for producing parts or devices having features or tolerances at the microscale. Exemplary techniques include, but are not limited to, lithography.

[0017] As used herein, a "microdevice" refers to any object or device having dimensions on the microscale, such as between 1 micron and 1000 microns, between 1 micron and 500 microns, between 1 micron and 250 microns, or between 1 micron and 100 microns.

[0018] The term "diameter" is recognized in the art and is used herein to refer to either the physical diameter or the hydrodynamic diameter. The diameter of an emulsion typically refers to the hydrodynamic diameter. The diameter of a capsule may refer to the physical diameter in a hydrated state, both for spherical and non-spherical shapes. The diameter of particles, colloids and cells enclosed inside a capsule refers to the physical diameter in a hydrated state. As used herein, the diameter of a non-spherical particle or capsule may refer to the maximum linear distance between two points on the surface of the particle. When referring to multiple particles or capsules, the diameter of the particle or capsule typically refers to the average diameter of the particle or capsule. The diameter of a particle or colloid can be measured using a variety of techniques, including, but not limited to, optical or electron microscopy, and dynamic light scattering.

[0019] The term "biocompatible" as used herein refers to one or more materials that are not themselves toxic to a host (e.g., an animal or human) and do not degrade (if the material degrades) at a rate that produces monomeric or oligomeric subunits or other by-products in toxic concentrations in the host.

[0020] The term "biodegradable" as used herein refers to the breaking down or disintegration of a material into its component subunits, or the digestion of the material into smaller (e.g., non-polymeric) subunits, e.g., by biochemical processes.

[0021] "Microspheres," "microparticles," or "microcapsules" are art-recognized and include substantially spherical solid or semi-solid structures formed, for example, from biocompatible polymers such as the present compositions, having sizes ranging from about 1 or more to about 1000 microns, such as 1 micron to 500 microns, 1 micron to 250 microns, or 1 micron to 100 microns. The term "microparticle" is also art-recognized and includes microspheres and microcapsules, as well as structures that cannot be easily classified into either of the above two categories, all of whose dimensions average less than about 1000 microns. Microparticles may be spherical or non-spherical and may have any regular or irregular shape. When the structures are less than about 1 micron in diameter, the corresponding art-recognized terms "nanospheres," "nanocapsules," and "nanoparticles" may be used.

[0022] As used herein, "narrow range release" generally means that the drug is released over a specific period of time, such as an hour, a few hours, a day, a week, a month, etc.

[0023] As used herein, "antigen" or "vaccine" refers to any molecule or entity that produces a specific immune response in a host organism, such as a mammal.

[0024] As used herein, "immune response" refers to a specific response to an antigen or vaccine that generates immunity in a host, such as a mammal, to any future challenge.

[0025] "Water soluble" generally refers to something that dissolves or dissolves in an aqueous environment.

[0026] "Emulsion" as used herein refers to separate phases of liquid homogeneously dispersed in a continuous phase of liquid.

[0027] As used herein, "hydrophilic" refers to a molecule that has a greater affinity for water compared to organic solvents and is therefore soluble in water. The hydrophilicity of a compound can be quantified by measuring the partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent such as octanol, ethyl acetate, methylene chloride, or methyl tert-butyl ether. A compound is considered to be hydrophilic if, after equilibration, there is a higher concentration of the compound in water than in the organic solvent.

[0028] As used herein, "hydrophobic" refers to a molecule that has a greater affinity for organic solvents compared to water, and is therefore soluble in organic solvents. The hydrophobicity of a compound can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent, such as octanol, ethyl acetate, methylene chloride, or methyl tert-butyl ether. A compound is considered to be hydrophobic if, after equilibration, there is a higher concentration of the compound in organic solvent than in water.

[0029] II. Preparations A. Polymer and Solvent Systems polymer Formulations that can be formed from microparticles, including those that are emulsion-based, or devices such as those prepared by micromolding, are formed from polymers. Antigens can be dispersed or encapsulated by polymers. In one embodiment, the device contains a core that contains only one or more vaccines or antigens and stabilizers, and a shell or particle wall that contains only one or more biodegradable polymers, with or without additives. Polymers without antigens can be used to seal or separate areas of the formulation from other areas and release at different rates.

[0030] The polymer must be biocompatible and processable under conditions and with reagents that preserve the antigen. The formulation can be made of hydrophilic polymers, hydrophobic polymers, amphiphilic polymers, or mixtures thereof. The formulation can contain one or more hydrophilic polymers.

[0031] Hydrophilic polymers include cellulosic polymers such as starch and polysaccharides; hydrophilic polypeptides; poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO); poly(oxyethylated polyols); poly(olefinic alcohols); polyvinylpyrrolidone; poly(hydroxyalkyl methacrylamide); poly(hydroxyalkyl methacrylates); poly(saccharides); poly(hydroxy acids); poly(vinyl alcohols), and copolymers thereof.

[0032] Examples of hydrophobic polymers include polyhydroxy acids, such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid); polyhydroxyalkanoates, such as poly 3-hydroxybutyrate or poly 4-hydroxybutyrate; polycaprolactones; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactone); polycarbonates, such as tyrosine polycarbonate; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); Examples of the hydrophobic polymer include polyesteramides, polyesters, poly(dioxanone), poly(alkylene alkylates), hydrophobic polyethers, polyurethanes, polyetheresters, polyacetals, polycyanoacrylates, polyacrylates, polymethylmethacrylates, polysiloxanes, poly(oxyethylene) / poly(oxypropylene) copolymers, polyketals, polyphosphates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(maleic acid), and copolymers thereof. In certain embodiments, the hydrophobic polymer is an aliphatic polyester. In preferred embodiments, the hydrophobic polymer is poly(lactic acid), poly(glycolic acid), or poly(lactic-co-glycolic acid).

[0033] The formulation can contain one or more biodegradable polymers.

[0034] Biodegradable polymers may include polymers that are insoluble or poorly soluble in water and are converted chemically or enzymatically to water-soluble materials within the body. Biodegradable polymers may include soluble polymers crosslinked with hydrolyzable crosslinking groups that render the crosslinked polymers insoluble or poorly soluble in water.

[0035] Biodegradable polymers include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidones, polyglycolides, polysiloxanes, polyurethanes and their copolymers, alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, polymers of acrylic and methacrylic acid esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose acetate terephthalate ... Examples of suitable polyvinyl chloride copolymers include cellulose triacetate, cellulose sulfate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohol), poly(vinyl acetate), polyvinyl chloride polystyrene and polyvinylpyrrolidone, derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof. Exemplary biodegradable polymers include polyesters, poly(orthoesters), poly(ethyleneimines), poly(caprolactones), poly(hydroxybutyrates), poly(hydroxyvalerates), polyanhydrides, poly(acrylic acids), polyglycolides, poly(urethanes), polycarbonates, polyphosphates, polyphosphazenes, derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof.

[0036] The amphiphilic polymer may be a polymer containing a hydrophobic polymer block and a hydrophilic polymer block. The hydrophobic polymer block may contain one or more of the above hydrophobic polymers or their derivatives or copolymers. The hydrophilic polymer block may contain one or more of the above hydrophilic polymers or their derivatives or copolymers.

[0037] In particularly preferred embodiments, the microparticles contain biodegradable polyesters or polyanhydrides, such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid). The microparticles can contain one or more of the following polyesters: homopolymers containing glycolic acid units (referred to herein as "PGA"), and lactic acid units such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide (collectively referred to herein as "PLA"), and caprolactone units such as poly(ε-caprolactone) (collectively referred to herein as "PCL"); and copolymers containing lactic acid and glycolic acid units, such as various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide) characterized by the ratio of lactic acid:glycolic acid (collectively referred to herein as "PLGA"); and polyacrylates, and derivatives thereof. Exemplary polymers also include copolymers of polyethylene glycol (PEG) with the polyesters described above, such as various forms of PLGA-PEG or PLA-PEG copolymers (collectively referred to herein as "PEGylated polymers"). In certain embodiments, the PEG region can be covalently attached to the polymer by a cleavable linker, resulting in a "PEGylated polymer."

[0038] The formulation can contain one polymer or a mixture of two or more polymers. The microparticles may contain other entities such as stabilizers, surfactants, or lipids.

[0039] solvent Since some residues are always present in polymer formulations, the solvent must be biocompatible. Representative polymer solvents include organic solvents such as chloroform, dichloromethane, tetrafluoroethylene, and acyl acetate. Antigens can be dissolved in aqueous or water-miscible solvents such as acetone, ethanol, methanol, isopropyl alcohol, and mixtures thereof.

[0040] B. Therapeutic, preventative, nutritional, and diagnostic agents Although described in the context of vaccine delivery, it will be appreciated that the formulations may be used to provide for the release of a variety of therapeutic, prophylactic, nutraceutical, and / or diagnostic agents. These agents may be small molecular weight drugs, proteins such as hormones or growth factors, immunomodulators, antibodies, nucleic acid molecules (DNA, RNA, microRNA, siRNA).

[0041] antigen infectious agent Antigens for delivery are killed or attenuated infectious agents such as bacteria, such as tetanus, viruses, such as hepatitis, influenza, and polio, and protozoa, such as Plasmodium (malaria) and Leishmania. Table 2 lists some vaccines whose antigens can be used in the disclosed formulations. Other antigens are antigenic proteins, or haptens, such as carbohydrates, or sugar antigens, that are effective as antigens for these infectious agents, as cancer antigens, or as immune stimulants.

[0042] Poliomyelitis (polio) is a highly contagious viral disease that invades the nervous system and can cause total paralysis in just a few hours. One in 200 infections leads to irreversible paralysis, which is usually limited to the legs. Of those who become paralyzed, 5%-10% die due to paralysis of the diaphragm. There is no treatment for polio. However, it can be prevented by vaccination.

[0043] Polio cases have declined by more than 99% since 1988, from an estimated 350,000 cases in over 125 endemic countries to only 223 reported cases in 2012. Currently, as of early 2013, only three countries in the world (Afghanistan, Nigeria, and Pakistan) are endemic to the disease. Despite aggressive vaccination efforts, polio has not been completely eradicated, and outbreaks still occur, especially in developing countries.

[0044] There are two types of vaccines that protect against polio: inactivated polio vaccine (IPV) and oral polio vaccine (OPV). IPV must be administered into the bloodstream to be effective. In contrast, OPV is effective by crossing the intestinal epithelium. OPV confers superior intestinal immunity, is easy to administer, and is inexpensive, but live poliovirus is shed by vaccinated individuals, which is a concern if the entire population is not vaccinated.

[0045] As such, the use of IPV is preferred.

[0046] cancer antigen Any protein produced in tumor cells that has an abnormal structure due to mutation can act as a tumor antigen. Such abnormal proteins are produced by the mutation of related genes. Mutations of protooncogenes and tumor suppressors that lead to the production of abnormal proteins are the cause of tumors, and therefore such abnormal proteins are called tumor-specific antigens. Examples of tumor-specific antigens include the abnormal products of ras and p53 genes. In contrast, mutations of other genes that are not involved in tumor formation can lead to the synthesis of abnormal proteins that are called tumor-associated antigens.

[0047] Proteins that are normally produced in very low amounts but whose production is dramatically increased in tumor cells trigger immune responses. An example of such a protein is the enzyme tyrosinase, which is required for melanin production. Normally, tyrosinase is produced in minute amounts, but its levels are greatly elevated in melanoma cells.

[0048] Oncofetal antigens are another important class of tumor antigens. Examples are alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA). These proteins are normally produced early in embryonic development and disappear by the time the immune system is fully developed. Therefore, self-tolerance to these antigens does not develop.

[0049] Abnormal proteins are also produced by cells infected with tumor viruses, such as Epstein-Barr virus ("EBV") and human papillomavirus ("HPV"). Cells infected with these viruses contain latent viral DNA that is transcribed, and the resulting proteins generate an immune response. In addition to proteins, other substances such as cell surface glycolipids and glycoproteins may also have abnormal structures in tumor cells and thus become targets of the immune system.

[0050] Many tumor antigens have the potential to be effective as tumor vaccines. In addition to alpha-fetoprotein (germ cell tumors, hepatocellular carcinoma) and carcinoembryonic antigen (intestinal, lung, breast cancer), examples of tumor antigens include CA-125 (ovarian cancer), MUC-1 (breast cancer), epithelial tumor antigen (breast cancer), and melanoma-associated antigen (malignant melanoma).

[0051] C. Stabilizers Antigen stability is defined as the maintenance of antigen structure during the formation of the vaccine formulation and at body temperature. As discussed below, the polymer composition, solvent selection, and processing conditions are critical to maintaining antigen stability.

[0052] Stabilizers may be added. Sugars are a typical group of stabilizers for proteins. Examples include simple sugars such as sucrose, fructose, mannitol, glucose, and trehalose, as well as more complex sugars. See Alcock et al., Long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass. Science Translational Medicine, 2(19):19-19ra12 (2010).

[0053] Antigen stabilization can be determined by antigen-specific ELISA in vitro and by measuring immune responses (e.g., IgG) in animals in vivo. Stability is evaluated during each step of the encapsulation and / or manufacturing process, at 25° C. (room temperature), during storage in humid / hot conditions, under physiological conditions (pH 7.2, 37° C.), and in vivo (animal models).

[0054] D. Rate of antigen uptake or completeness of release A gas-generated burst release system may allow for the immediate release of the encapsulated antigen. Pore-forming agents may also be utilized that are removed by leaching or lyophilization.

[0055] III. Method of manufacture It is essential that the methods used to manufacture the device maintain antigen stability both during processing and at body temperature, and minimize leakage after formulation and administration. Sterilization after formulation can typically be achieved by a combination of aseptic manufacturing conditions combined with methods such as gamma radiation.

[0056] A. Emulsion Standard techniques can be used to make the microparticles. A preferred technique is emulsification of a solution of the polymer in an organic solvent with an aqueous solution. Addition of the organic phase to the bulk of the non-solvent phase forms a spontaneous single emulsion, and the resulting solution is constantly stirred to allow the solvent to evaporate. The formation of microspheres occurs immediately. After stirring, the microspheres are washed and then dried.

[0057] The examples demonstrate the formation of microparticles using emulsification of polymers and antigens, either alone or in combination with stabilizers such as trehalose and sucrose.

[0058] B.3D printing 3D printing can increase the consistency of the microspheres, allowing for more uniform release, and can provide a means to create more complex devices, such as "microrods" with increased loading capacity, which can eliminate the need for simultaneous release from multiple microspheres and facilitate scale-up.

[0059] Three-dimensional (3D) printing is the process of creating a 3D object from a digital model. 3D printing is an additive process, in which successive layers of material are laid down in different shapes. After each layer is added, the "ink" is polymerized, typically by photopolymerization, and the process is repeated until the 3D object is created. Recent commercial availability and reduced costs make 3D printing of biomolecules, including vaccines and medicines, attractive for distribution of these compounds to developing countries. This eliminates the need to ship the final product to the country. Instead, 3D printers can print out the requested biomolecules from a simple computer program that can be brought to the site from anywhere in the world at the point of care.

[0060] The 3D printing workflow can be described in three sequential steps: 1) the powder delivery system platform is raised and the fabrication platform is lowered one layer; 2) a roller spreads the polymer powder into a thin layer; 3) a print head prints a liquid binder that bonds adjacent powder particles together. Billiet et al., Biomaterials, 33:6020-6041 (2012). For nanobiomaterial fabrication, two types of 3D printing techniques are mostly employed. One is inkjet printing using a typical printer. Marizza et al., Microelectrionic Engin. 111:391-395 (2013). The other is nanoimprint lithography.

[0061] Nanoimprint lithography (NIL) is a rapid and cost-effective technique for fabricating nanostructures. The NIL procedure involves stacking multiple layers of such structures on top of each other; i.e., the completed bilayer structure is covered with a spacer layer that is planarized using chemical-mechanical polishing so that a second layer can be fabricated on top. Liu et al., J. Nanomat. Aug. (2013).

[0062] Inkjet printing has been used to generate monodisperse PLGA particles. Bohmer et al., Colloids and Surfaces A: Physiochem. Eng. Aspects, 289:96-104 (2006). Briefly, droplets of a PLGA solution are printed with an inkjet nozzle immersed in an aqueous phase. This method produces microspheres with predictable and controllable sizes. Using this technique, paclitaxel-loaded monodisperse microspheres have been created. Radulecu et al., Digital Fabrication Sep:18-21 (2005). Modifications of this technique have been used to create multi-layered monodisperse microspheres. See Kim and Pack, BioMEMS and Biomedical Nanotechnology, 1:19-50 (2006). Using this technology, the thickness of the microcapsule shell can be varied from less than 2 microns to tens of microns while maintaining complete and well-concentrated core encapsulation for microcapsules approaching 50 microns in overall diameter.

[0063] Drug delivery rates from microspheres have been modified by providing uniform monodisperse microparticles, mixtures of microparticles of various sizes, and microparticles with different degradable layers. See Kim and Pack, BioMEMS and Biomedical Nanotechnology, 1:19-50 (2006).

[0064] Internet site store.makerbot.com / replicator2 (2013); Tekin et al., Inkjet printing as a deposition and patterning tool for polymers and inorganic particles.Soft Matter, 4:703-713(2008);Jang et al., Influence of fluid physical properties on ink-jet printability.Langmuir, 25:2629-2635(2009);Lan, Design and Fabrication of a Modular Multi-Material 3D Printer., M.Sc.Thesis:Massachusetts Institute of Technology, 2013; and the website imagexpert.com / site-new / pdf / IXjetXpert.pdf.(2013) for further information.

[0065] The examples demonstrate the preparation of microparticles using 3DP. The waveform was optimized to consistently jet monodisperse ink droplets. The applied voltage, the duration of the applied voltage, and the change in voltage over time (slope) are all parameters that must be optimized to jet high quality ink droplets. For example, the waveform was optimized for a solution of 5 w / v% 31k (average molecular weight) PLGA in 1,4-dioxane. The waveform was optimized using a JetXpert imaging system, and the ink and waveform were then transferred to a composite inkjet 3D printer. JetXpert imaging was performed with a constant pressure waveform. In one embodiment, 5 w / v% 31k PLGA / 1,4-dioxane (Z=5.3, η=6.08 mPascal sec) was used. The waveform was optimized for this specific solution. In another embodiment, 15 w / v% 12k PLGA / 1,2-dichloroethane (Z=5.8, η=6.24 mPascal sec) was used. Most nozzles showed optimal jetting as in the first embodiment, but very few nozzles produced droplet trains containing satellite droplets. In the third embodiment, 15 w / v% 12k PLGA / chloroform (Z=5.8, η=5.99 mPascal sec) was used (Table 1). When more volatile solvents (chloroform or acetone) were used, most nozzles fired more than one droplet continuously. In general, for a given optimized waveform and similar fluid properties, inks made with more volatile solvents resulted in inconsistent jetting, and printhead nozzles clogged during printing (observed when using 1,2-dichloroethane or chloroform). To prevent nozzle clogging, inks can be made with 1,4-dioxane and DMF. Printing with these solvents requires longer drying times between layers to prevent the structure from morphing. For a given polymer, increasing the polymer concentration in the ink increased the ink viscosity as determined by GPC. For a given polymer concentration, increasing the molecular weight range of the polymer increased the ink viscosity, and the observed changes in ink density and surface tension after polymer addition were negligible compared to the viscosity change.

[0066] Figures 11A and 11B are schematics of particles made by emulsion (Figure 11A) and by 3D printing (Figure 11B), as well as the release of proteins from emulsion particles (Figure 11C) and idealized 3DP particles (Figure 11D). These show differences in the resulting structures, which also alter the release kinetics.

[0067] 12A-12D are schematics of the 3D printing process: creating a PLGA particle structure (FIGS. 12A and 24A), loading the particles with drug or protein (FIGS. 12B and 24B), drying the drug or protein (FIG. 12C), and encapsulating the particles (FIGS. 12D and 24C). The schematics show a cube-shaped structure filled with vaccine, but it will be understood that the mold may be utilized to provide any shape, and may be filled in layers or in more complex patterns. Table 1: Solvents for 3DP [Table 1]

[0068] The waveform parameters can be optimized to produce uniform, single droplets of "ink" (polymer solution) from the piezoelectric nozzle jet during printing (Figure 13). Drop size is a function of various optimized parameters, including applied voltage, voltage slope, polymer and solvent selection and concentration.

[0069] C. Micromolding Park et al., Biomed.Microdevices, 9:223-234 (2007) describes the use of micromolding to fabricate polymer microstructures with sophisticated designs. The micromolds were filled with polymer microparticles to generate multi-material microstructures with complex geometries and fabricated using mild processing conditions. Typically, oil-water, double emulsion systems; spray drying; supercritical conditioning; and milling methods are used to prepare these microparticles. In a preferred embodiment, the micromolds can be prepared by photolithographically creating a female master mold made of photoresist, molding a male master structure from the female master mold in polydimethylsiloxane (PDMS), and molding a female replica mold made of PDMS from the male master structure. The polymer microparticles can be micromolded using temperature / pressure methods and / or from solvents.

[0070] Using spray drying and emulsion techniques, polymer microparticles with sizes ranging from 1 to 30 μm were fabricated from PLA, PGA and PLGA. These polymer microparticles were filled into PDMS micromolds at room temperature and then melted or bonded together, for example by ultrasonically welding the microparticles together in the mold, while at the same time maintaining the voids inherent in the packing structure. Multi-layered microstructures were fabricated to have polymers of different compositions, and the encapsulated compounds were located in different regions of the microstructure. The mold was filled with solid polymer microparticles instead of polymer melt to replicate microstructures with complex geometric forms and composed of multiple materials using mild processing conditions. The microparticles can easily flow into the cavities of the micromold at room temperature and low pressure, which makes it easy to create microstructures with high aspect ratios. Furthermore, the polymer microparticles can encapsulate chemical compounds such as drugs, and the mold can be filled in successive layers to accommodate compositions of multiple materials. After the mold is filled, the final microstructure can be created by welding the microparticles within the mold using plastic welding techniques, including heat and ultrasonic welding, and solvent and gas welding.

[0071] These same techniques can be used to formulate vaccine formulations once the polymeric materials and conditions required to obtain a narrow time period of release at a particular point after administration have been identified.

[0072] IV. Method of Administration Vaccine formulations are administered to individuals in need of vaccination as dosage formulations containing an effective amount of one or more antigens, released on a schedule that elicits protection against the source of the antigen.

[0073] The microparticles or microcapsules can be administered by injection, preferably subcutaneously or intramuscularly, for example subcutaneously on the back of the upper arm, or to a mucosal surface (oral, intranasal, via the pulmonary route, or other orifice), although injection is preferred if release is to occur over an extended period of more than several days.

[0074] The dosage form is designed to release a bolus of antigen at the time of administration. This can be accomplished by administering a solution or dispersion of antigen in combination with a vaccine formulation that provides multiple releases at later times, or the device can be formulated to provide an initial bolus and subsequent release(s). Representative vaccines are shown in Table 2: [Table 2]

[0075] Further vaccines of great interest in Third World countries include polio and smallpox, and the polio vaccine will be used below as an example vaccine for this application.

[0076] IPV Vaccine SSI is an inactivated vaccine used for prophylactic vaccination against paralytic poliomyelitis. IPV Vaccine SSI contains inactivated poliovirus types 1, 2 and 3 grown in Vero cells. Serving Size (0.5 mL): Inactivated poliovirus type 1 (Bruenhilde) 40 D antigenic units; Inactivated poliovirus type 2 (MEF-1) 8D antigenic units; Inactivated poliovirus type 3 (Saukett) 32D antigenic units; 199 medium up to 0.5 mL.

[0077] The vaccine is produced without serum and trypsin and contains no preservatives or adjuvants. No antibiotics are used in the production. The IPV vaccine SSI contains traces of residual formaldehyde. It is manufactured in Denmark by Statens Serum It is manufactured by the Institute.

[0078] The IPV vaccine SSI is a solution for injection dispensed in single-dose vials. A series of three doses of 0.5 ml is administered for the initial vaccination.

[0079] For booster vaccination of previously primary vaccinated humans, one dose of 0.5 ml is administered at the earliest 6 months after the primary vaccination series. Administration of additional booster doses should be done according to national recommendations for polio immunization. The vaccine should be administered intramuscularly or subcutaneously. The vaccine should not be administered intravascularly. Age at first dose should be at least 6 weeks, and the primary vaccination series should include at least 3 immunizations at least 4 weeks apart. Most countries give IPV using the same schedule as the DPT vaccine (typically at 2 months, 4 months, and 6 months of age).

[0080] The immunogenicity and safety of IPV vaccine SSIs have been investigated in several clinical trials, including clinical trials with combination vaccines for pediatric use. In addition to IPV, these trials included vaccine antigens against tetanus, diphtheria, pertussis, and Haemophilus influenzae type b.

[0081] If immunization begins at 2 months of age, completion of a primary vaccination series of three immunizations spaced at least one month apart can be expected to result in seroconversion to all three types of poliovirus one month after the second immunization. Seroconversion rates of 89% to 99% have been demonstrated if immunization begins before 2 months of age and as early as 6 weeks of age. Thus, booster doses at 9 months of age or in the second year of life should be considered with such a schedule.

[0082] IPV vaccine SSI can be used for revaccination of infants, preschoolers, and adults who were immunized with IPV or OPV. IPV vaccine SSI can be used in a combined IPV / OPV schedule using 1-3 doses of IPV followed by 1-3 doses of OPV. It is recommended that IPV be administered before the first dose of OPV. In combined IPV / OPV schedules, persistence of protective antibodies after primary vaccination has been shown to last for at least 20 years. Recommended doses for sustained protection SSI in IPV-only schedule: D antigen type 1-40DU / ml D antigen type 2-8DU / ml D antigen type 3 - 32DU / ml 10x trivalent IPV: D antigen type 1-327DU / ml D antigen type 2-70DU / ml D antigen type 3-279DU / ml

[0083] The present invention will be further understood by the following non-limiting examples. EXAMPLES

[0084] The following examples describe various polymer-drug or polymer-antigen formulations for controlled release of drug / antigen. Controlled release relies on polymer degradation to achieve multiple bursts of drug / antigen release over time after a single injection. The immunogenicity of the formulations after the burst of drug / antigen release based on polymer degradation and the sustained increase in anti-antigen antibody titers are also presented. Methods for making the formulations are also described, including the incorporation of antigen into the polymer matrix via spontaneously forming emulsion, or the encapsulation of antigen within a polymer shell via 3D printing or micromolding. Studies to improve antigen stability, polymer matrix stability, and formulation stability are also presented, which can result in formulations with desired release characteristics and immunogenicity.

[0085] Example 1: Selection of polymers and solvents for separate release of vaccines material and method Polymer type-PLGA, PLLA; Polymer molecular weight-9.5k, 20k, 31k, 46k Drug loading- 0.5, 3, 5%; Excipients- trehalose, sucrose Encapsulation - Naturally Forming Emulsions In this process, CH is used as the organic phase. 2 Cl 2 :TFE::4:1.(CH 2 Cl 2 -dichloromethane (TFE-trifluoroethanol) was used with poly(vinyl alcohol) ("PVA") to encapsulate 5%, 3% or 0.5% bovine serum albumin ("BSA") or inactivated poliovirus ("IPV") into the polymer. Addition of the organic phase to the bulk non-solvent phase forms a spontaneously forming single emulsion, and the resulting solution is constantly stirred to evaporate the solvent. Microspheres are formed immediately. After stirring, the microspheres are washed and then dried. See Jaklenec et al., Sequential release of bioactive IGF-I and TGF-β1 from PLGA microsphere-based scaffolds. Biomaterials, 29(10):1518-1525 (April 2008).

[0086] In vitro release studies were performed with 10 mg of microspheres suspended in 1 ml of PBS buffer (pH 7.2) at 37° C. Time points used were days 1, 4, 7 and weekly thereafter. At each time point, vials were centrifuged, the supernatant removed and assayed, 1 ml of fresh PBS was added, and the pelleted microspheres were resuspended.

[0087] result FIG. 21A is a graph of the release over time (weeks) of the model protein bovine serum albumin ("BSA") from carboxyl-derivatized PLGA (50:50) (20 kD) for 5% BSA, 3% BSA, and 0.5% BSA.

[0088] FIG. 21D is a graph of the release of bovine serum albumin over time (weeks) from carboxyl-derivatized PLGA (50:50) (9.5 kD) with 5% BSA, 3% BSA, and 0.5% BSA.

[0089] These figures show the release from the same polymer, but with different molecular weights. This difference alone dramatically changed the release profile.

[0090] 3A and 3B are graphs of the release of bovine serum albumin from PLLA (50 kD) over time (weeks) for 5% BSA and 0.5% BSA.

[0091] 4A and 4B are graphs of the release of bovine serum albumin from PLLA (100 kD) over time (weeks) for 5% BSA and 0.5% BSA.

[0092] 5A and 5B are graphs of the release of bovine serum albumin from PLLA (300 kD) over time (weeks) for 5% BSA and 0.5% BSA.

[0093] The only difference between these three graphs is the molecular weight. However, in none of the cases was there a substantially different release period similar to the range in FIG.

[0094] 6A and 6B are graphs of the release of bovine serum albumin from P(d,l)LA (20 kD) over time (weeks) with 5% BSA and 0.5% BSA. Results are similar to Figures 3-5 and 21.

[0095] The best results were obtained using PLGA-COOH (50:50), which is summarized in Figures 7A and 7B for the PLGA formulation (Figure 7A) compared to bolus injection (Figure 7B).

[0096] Details of the formulations used in Figure 7A are presented in Table 3 (see also Tables 4 and 5). In Table 3, the difference between F3 and F7 is the molecular weight of the polymer. F5 has a high loading of BSA. F3 and F7 have about three equal doses, but F5 has a larger initial dose followed by two smaller doses, which is very similar to an initial bolus with two booster shots. The formulation is injected into the animals on day 0 (single injection), while a bolus control is injected three times during the course of the study to mimic the formulation release kinetics. Negative controls include blank microspheres for the formulation, and saline for the bolus injection. The injection volume was 200 μl (maximum volume for subcutaneous (SC) injection in mice). Two injections of 200 μl injections (one per leg) were performed. The maximum injectable microsphere concentration was 50 mg / ml. Table 3. Amount of BSA released (μg) in the first, second and third peaks after injection of F3, F5 and F7 formulations. [Table 3]

[0097] 8A and 8B are graphs of the release of 0.5% bovine serum albumin over time (weeks) compared to the release of 0.5% ovalbumin from carboxyl-derivatized PLGA (50:50) (20 kD).

[0098] 9A and 9B are graphs of the release of 5% bovine serum albumin over time (weeks) compared to the release of 5% ovalbumin from carboxyl-derivatized PLGA (50:50) (31 kD).

[0099] 10A and 10B are graphs of the release over time (weeks) of 0.5% bovine serum albumin compared to the release of 0.5% ovalbumin from carboxyl-derivatized PLGA (50:50) (31 kD).

[0100] All of these show excellent results that vary only with the molecular weight or percent loading of the polymer.

[0101] Example 2: Predicted release profiles from microparticles made by emulsion compared to microparticles made by 3DP Particles fabricated by computer-controlled inkjet 3D printing can be made with identical micrometer-scale dimensions, drug loading, and spatial location of the drug within the polymer microstructure. The main difference between the two methods is that emulsion-based particles (left) are matrix-based and the drug is homogenously distributed throughout the particle. 3D or micromolded particles are shown on the right. There is a clear separation of the vaccine and polymer, where the vaccine is present in the core and the polymer is only in the shell. These distinctions allow for unique control of the release kinetics between the two particle types. Micromolded / 3D printed particles also allow control of the core size, allowing for a higher degree of loading.

[0102] The release graph in FIG.

[0103] In the schematic of Figure 11E, drugs or vaccines are encapsulated in polymer microspheres and dispersed throughout the polymer matrix (A). Once hydrated, any drug on the surface is released immediately (B), thus causing an initial burst. Following this event, the microspheres have residual pores through which the drug can slowly diffuse, causing a second burst (C). Finally, when the polymer bulk erodes, the remainder of the drug is released in a third burst due to significant molecular weight degradation (D).

[0104] Example 3: Selection of piezoelectric jetting parameters to produce microparticles material and method optimization: The pressure applied through the nozzle to eject uniform, single droplets of PLGA (Waveform) is corrected. Optimize the viscosity of the PLGA solution (Z number). The viscosity must be low enough to be effectively sprayed through a nozzle. Make the viscosity high enough to solidify on the substrate.

[0105] Tests have shown that proper drop formation in piezoelectric drop-on-demand (DOD) inkjet printing can be described using the dimensionless Z-number, which is determined by the ink's fluid properties (surface tension, density, and viscosity) and the size of the printhead nozzle.

[0106] A Z number range can be defined to determine the printability of an ink with a particular waveform. Characterize PLGA solutions using Z numbers:

[0107] Solvents are selected based on PLGA solubility and physical properties (ie, vapor pressure and boiling point).

[0108] Once the optimal waveform is determined for one PLGA solution ink, the same waveform can be used to print with PLGA of different molecular weights by using PLGA solutions with Z numbers within a certain range.

[0109] result The waveforms for jetting the BSA and IPV solutions were optimized as shown in Figure 13. This allows the polymer printer particles to be loaded with the vaccine.

[0110] Example 4: Studies to minimize IPV D antigen loss and increase stability during lyophilization and encapsulation To prevent loss of D antigen due to lyophilization, studies were carried out to optimize methods and reagents for concentrating IPV prior to encapsulation. Methods that can be used include centrifugal filtration and dialysis.

[0111] Excipients that may reduce D antigen loss during the emulsion process were tested. Some success was achieved using BSA. The tests utilized sugar. Varying sugar concentrations were tested to form sugar glasses. Varying humidity conditions were also tested for drying.

[0112] Comparisons were also made with other solvent systems for the process of spontaneously forming emulsions. In early studies, chloroform / acetone was used for the organic phase. In subsequent studies, ethyl acetate was used instead of dichloromethane (DCM).

[0113] material and method Alcock et al. reported that viruses incorporated into sugar glasses exhibited long-term solid-state stability. (Alcock et al., Science Translational Medicine, 2(19):19-19ra12 (2010)) IPV stability on 3D printed substrates was tested in a similar manner. IPV solutions containing co-dissolved sugars (sucrose and trehalose) were deposited onto scaled-up PLA structures printed using a MakerBot Replicator2. Drying of the IPV / sugar solutions was performed at ambient humidity (approximately 20% humidity), which was higher than the humidity used by Alcock et al. (approximately 10%).

[0114] result Figures 14A, 14B and 14C are graphs of the percent of D antigen (IPV types I, II and III) retained after lyophilization with the sugar excipients 1 M trehalose, 1 M sucrose and 3 M sucrose followed by incubation at 4°C, 25°C or 37°C.

[0115] The results demonstrate that sugars significantly increased the stability of IPV, and there was only a small difference in stability at 4° C. and 25° C., but the stability was lower at 37° C. The results also showed that the stability differed depending on the IPV type.

[0116] Figures 15A and 15B are graphs of the percent of D antigen (IPV types I, II, and III) retained after lyophilization with 0, 0.5, 0.75, 1, or 1.25 M trehalose and subsequent incubation at 4° C. (Figure 15A) or 25° C. (Figure 15B). Best results were obtained with 0.5 M and 0.75 M trehalose.

[0117] Figures 16A and 16B are graphs of the percent of D antigens (IPV types I, II, and III) retained after mixing with solvents until the solvent evaporated using tetrafluoroethylene ("TFE"), dichloromethane ("DCM"), or TFE and DCM as the solvent, without a sugar excipient (Figure 16A) or with 0.75 M trehalose as the excipient (Figure 16B). DCM was statistically significantly better. IPV was not stable when exposed to organic solvents during the encapsulation process, even when trehalose was used as an excipient.

[0118] Figure 17A is a graph of the percent of D antigens (types I, II, and III IPVs) retained after IVPs with 0M sugar, 0.5M trehalose, or 0.5M trehalose·sucrose were printed onto 3D-printed PLA substrates and dried at 25°C and 22% relative humidity. Figure 17B is a graph of the percent of D antigens (types I, II, and III IPVs) retained after IVPs with 0.25M trehalose or 0.5M trehalose·sucrose were printed onto 3D-printed PLA substrates and dried at 25°C and 10.7% relative humidity.

[0119] Alcock et al. dried the virus / sugar solution onto a substrate under controlled conditions (20-25°C, 2-10% relative humidity). Drying for Figure 17A involved IPV / sugar solution onto a PLA substrate in a ventilated cell culture hood (20-25°C, 22.9% relative humidity). Drying for Figure 17B involved IPV / sugar solution onto a PLA substrate in a desiccator (20-25°C, 10.7% relative humidity). Stability was good with respect to room temperature / humidity (all three IPV types retained >50% D antigen).

[0120] Figures 18A and 18B are graphs of the percent of D antigen retained after lyophilization with 0.5 or 0.75 M trehalose followed by incubation at 25°C (Figure 18A), or after lyophilization with pipetted or jetted 0.5 M trehalose·sucrose followed by drying at 25°C and 10.7% relative humidity (Figure 18B).

[0121] These studies demonstrate that the best results were obtained by storing 3D-printed PLA particles containing freeze-dried IPV at room temperature and humidity (approximately 20% relative humidity). The studies also demonstrated that jetting IPV did not significantly reduce IPV stability compared to freeze-drying IPV.

[0122] Example 5: Short-term in vivo immunogenicity of BSA-containing PLGA formulations Immune responses, expressed as anti-BSA IgG (antibody) titers, were measured 1, 2, and 4 weeks after injection of formulations F3, F5, and F7, and are presented in Table 3 above and Table 4 below. Formulations F4 and F8 were empty microspheres (no drug) and were used as negative controls (see Table 4).

[0123] The results are presented in Figures 19A, 19B, and 19C. Antibody titers (log2) are plotted for the various groups at weeks 1, 2, and 4. The negative controls are all zero, and the microsphere formulations produce equivalent or stronger responses compared to the bolus control.

[0124] Example 6: Long-term in vivo immunogenicity of BSA-containing PLGA microspheres material and method material Poly(D,L-lactic-co-glycolic acid) (PLGA Resomer® RG502H, RG503H, RG504H, and RG752H) and BSA were purchased from Sigma-Aldrich (St. Louis, MO). Poly(vinyl alcohol) (PVA, molecular weight = 25,000) was purchased from Polysciences, Inc. (Warrington, PA). Dichloromethane (DCM) and 2,2,2-trifluoroethanol (TFE) used in this study were reagent grade.

[0125] Microsphere fabrication Sixteen formulations of PLGA microspheres containing BSA (Table 4) were fabricated using the spontaneously forming single emulsion method (Fu et al., J Pharm Sci, 92:1582-1591, 2003; and Jaklenec et al., Biomaterials, 29:185-192, 2008). Briefly, 200 mg of PLGA was dissolved in 10 mL of 4:1 DCM:TFE and mixed with 300 μL of BSA (in water). Upon mixing, a clear single-phase solution was formed, which was then added to 200 mL of 5 (w / v)% PVA (in water). The spontaneously formed emulsion was stirred for 3 hours at room temperature. The particles were then collected by centrifugation, washed five times with water, and lyophilized. When preparing for in vivo use, the organic phase and BSA solution were filtered through a 0.2 μm polytetrafluoroethylene filter (Whatman, Little Chalfont, UK) and combined in a sterile laminar flow hood.

[0126] Table 4. Microsphere formulations and size characterization [Table 4]

[0127] Characterization of microspheres Microsphere size distribution was determined using a Multisizer3 Coulter Counter. Histograms were generated using a bin size of 0.39 μm and smoothed using a centered moving average with a window size of ±5 bins. Scanning electron microscope (SEM) images were collected at an accelerating voltage of 5 kV using a JSM-5600LV SEM (JEOL, Tokyo, Japan). Prior to imaging, samples were coated with Au / Pd to prevent surface charging using a Hummer 6.2 sputtering system (Anatech, Battle Creek, MI).

[0128] In vitro BSA release 10 mg of microspheres were dispersed in 1 mL of phosphate-buffered saline (PBS) in a capped tube and incubated at 37°C on a rotating platform. At each time point (day 1, then weekly for weeks 1–13), samples were centrifuged at 1500 relative centrifugal force (RCF) for 5 min, after which the supernatant was collected. Samples were then resuspended in fresh PBS and returned to the incubator for sampling at subsequent time points. BSA release from the microspheres was quantified using a bicinchoninic acid (BCA) assay and normalized to the total amount released by the end of the study. Samples were run in triplicate, and data were reported as the mean ± standard deviation.

[0129] In vivo administration of BSA microspheres All animal studies were approved by the MIT Animal Care Committee. Briefly, 6-8 week old female BALB / c mice were injected with (1) BSA-loaded microspheres, (2) unloaded microspheres, (3) bolus BSA, or (4) saline alone. The first two groups of mice were injected only once, whereas mice receiving bolus BSA or saline alone were injected again at weeks 4 and 8 to match the amount and timing of BSA release from PLGA microspheres in vitro. Samples were dissolved or suspended (where applicable) in 200 μl saline and injected subcutaneously in each hind limb with a total volume of 400 μl. Table 5 contains the exact dosing schedule for each group. At weeks 0, 1, 2, 4, 6, 8, and 10, 100 μl of blood was collected submandibularly and, after clotting, centrifuged at 2000 RCF for 10 min at 4°C to separate serum.

[0130] Table 5. Dosing regimens for in vivo BSA administration [Table 5]

[0131] Immunogenicity of BSA released from PLGA microspheres Serum antibody titers against BSA were determined using an end-point enzyme-linked immunosorbent assay (ELISA). 96-well Maxisorp ELISA plates (Thermo Fisher Scientific, Waltham, MA) were coated overnight at 4°C with 100 μL of a 100 μg / ml solution of BSA in 0.1 M sodium bicarbonate. The plates were then washed three times with PBS containing 0.05% Tween 20 (PBST) and then incubated for 2 hours at 37°C in 5% nonfat milk in PBST as a blocking agent. After another series of three washes with PBST, mouse serum samples were added in four-fold serial dilutions and incubated for 2 hours at 37°C. As the test progressed, the degree of serum dilution was increased and the titers increased. Plates were then washed five times with PBST and incubated for 2 h at 37°C with horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Southern Biotechnology Associates, Birmingham, Alabama) diluted 1:1000 in blocking buffer. Plates were washed another five times with PBST and developed using 100 μL of p-nitrophenyl phosphate solution, prepared from a tablet dissolved in 1× diethanolamine buffer from an alkaline phosphate substrate kit (Bio-Rad, Hercules, California). After 10 min, the reaction was stopped by adding 100 μL of 0.4 M sodium hydroxide to each well, and absorbance values ​​were read at 405 nm using a Tecan Infinite M200 Pro microplate reader (Maennedorf, Switzerland). Titers were reported as the reciprocal of the highest serum dilution that produced an absorbance greater than twice the background value.

[0132] statistical analysis All data in Table 4 and Figure 20 are reported as mean ± standard deviation. In vitro studies were performed with triplicate experiments, while in vivo studies were performed with 10 replicates for all groups at all time points, with the exception of Formulation C at week 10, where 9 replicates were used due to insufficient blood volume from one animal. Time-matched antibody titers were compared using one-way ANOVA statistical analysis for the three low dose groups, and unpaired two-tailed Student's t-tests for comparison of high dose microspheres and bolus injections. Comparison of peak antibody titers was performed at a significance level of 0.05 using unpaired two-tailed Student's t-tests with Holm-Bonferroni correction to attenuate the effect of multiple comparisons.

[0133] result Characterization of BSA-containing PLGA microspheres All 16 formulations of PLGA and BSA produced spherical microparticles with a wide distribution of particle sizes (Table 4). The size and shape of formulations C, G, and E used in the in vivo studies were representative of all formulations. Formulation C produced microspheres that were 10.3±6.2 μm in diameter, but due to a cubic relationship between volume and diameter, particles smaller than 10.3 μm contained only 4.2% of the antigen loading, assuming a homogeneous distribution of BSA in PLGA (Narasimhan et al., J Control Release, 47:13-20, 1997). The larger particles contained the majority of the antigen, with 90% of the volume contained in particles larger than 22.1 μm for formulation C. Formulations G and E demonstrated similar characteristics with particle diameters of 12.1±8.2 and 8.6±6.7 μm, respectively, but 90% of the particle volume was contained in particles larger than 23.1 and 21.4 μm, respectively. Histograms of microsphere diameter and volume distribution can be seen in Figures 20A-20F. Particles at the larger end of the distribution also contributed substantially to the surface area effect, as 50% of the cumulative particle surface area was present in particles larger than 23.67, 29.54, and 27.58 μm in diameter for formulations C, G, and E, respectively.

[0134] In vitro release of BSA from PLGA microspheres In vitro release kinetics from PLGA microspheres was determined by BCA assay and expressed as a percentage of total BSA released over the duration of the experiment. BSA release and particle degradation occurred more rapidly in PLGA microspheres fabricated using any of the three polymers in a 50:50 ratio of lactic acid to glycolic acid compared to the 75:25 ratio. All microsphere formulations produced using the 50:50 ratio of PLGA degraded within 14 weeks, whereas the 75:25 PLGA degraded at 22 weeks. The timing of BSA release appeared to be more dependent on the polymer type than on the BSA loading, although in most cases the loading primarily affected the size of the burst. As shown in Figure 21A, low molecular weight (7-17 kDa) PLGA released in three separate bursts over the course of 8 weeks and was completely degraded by the 14th week. Intermediate molecular weight (24-38 kDa) PLGA also showed three bursts spread over 9-12 weeks, depending on BSA loading, and degraded by week 14 (Figure 21B). The highest molecular weight (38-54 kDa) PLGA released BSA over 9-12 weeks with notable bursts at day 1 and week 8, but with more continuous release kinetics between 3% and 5% BSA-loaded microspheres (Figure 21C). Microspheres with higher lactate content (75:25) made from low molecular weight (4-15 kDa) PLGA degraded over 22 weeks and showed a gradual, semi-continuous release after an initial burst (Figure 21D).

[0135] Of the 16 in vitro formulations, formulations C, G, and E were selected for subsequent in vivo testing based on their in vitro release kinetics. Formulation C microspheres were made using low molecular weight PLGA (7-17 kDa) loaded with 0.5% BSA, formulation G microspheres were made with slightly higher molecular weight PLGA (24-38 kDa) and 0.5% BSA, and formulation E microspheres were made with the same molecular weight PLGA as formulation G but with a higher (5%) BSA loading. BSA release from formulation C was characterized by three distinct peaks, with 33.4±5.1% of the total BSA released at day 1, 27.2±4.3% released at week 4, and 32.1±5.2% released over the 6 and 7 week time points, respectively (FIG. 21A). Minimal BSA release was observed at weeks 1, 2, 4, and 5, or after the 7th week, and the microspheres were completely degraded by week 10, as evidenced by complete dissolution of the particles.

[0136] Formulation G microspheres also feature BSA release in three bursts (FIG. 21B), but spread over a longer time frame. In addition, total microsphere degradation was observed after 14 weeks, rather than 10 weeks for Formulation C. The first BSA burst from Formulation G was observed at the 1 day time point, with 28.0±5.7% of the total BSA released. This was followed by a second burst of 12.9±2.9% at 4 weeks, and the burst extended from 8 to 11 weeks, during which a cumulative 43.8±2.0% of BSA was released. Little or no BSA was observed in the release medium at any other time point throughout the complete degradation of the particles.

[0137] Formulation E microspheres released 63.7±7.3% of their BSA on day 1, which was the largest initial burst in terms of both total amount and total percentage of any formulation (FIG. 21C). The only time points with substantial BSA release after this initial burst were weeks 3 and 8, when 8.9±0.9% and 8.8±2.1% of the total load were released, respectively. Similar to formulation G, which used PLGA of the same molecular weight (24-38 kDa), formulation E was completely degraded by week 14.

[0138] Immunogenicity of BSA-containing PLGA microspheres Humoral immune responses to each microsphere formulation were compared to a positive control consisting of three bolus injections that matched the amount and timing of BSA released from the particles in vitro. Within an experimental group, a statistically significant increase in titer between successive weeks was used as evidence of release (one-sample paired t-test). Formulation C induced a significant increase in antibody titer compared to previous time points at weeks 1, 2, and 4 (p<0.05, p<0.0001, and p<0.001, respectively), then a significant decrease at weeks 6 and 8 (p<0.01 and p<0.05, respectively), before stabilizing at week 10 (Figure 22). Similarly, mice receiving formulation G showed a significant increase in titer at weeks 1, 2, and 4 (p<0.05, p<0.001, and p<0.01, respectively), remained stable at week 6, then dropped significantly by week 8 (p<0.05), before stabilizing again at week 10. Formulation E induced a similar response, with antibody titers increasing significantly at weeks 1, 2, and 4 (p<0.001 for all), plateauing at week 6, and then declining by the end of the study (p<0.05). Overall, the immune responses to all three microparticle formulations demonstrated a similar progression over time, with titers rising over the first four weeks and then slowly declining by week 10 (FIG. 22). However, the magnitude of antibody titers appeared to be highly dependent on BSA loading. Based on in vitro experiments, formulation E released approximately 13-fold more BSA than formulation C at the earliest time point (day 1) due to a large initial burst, and induced 13-fold higher antibody titers as well. This trend was also observed at the end of the study, as antibody titers induced by formulation E were 8-fold higher than those associated with formulation C after releasing 7-fold more BSA.

[0139] Antibody titers from animals receiving either of the BSA-loaded microsphere formulations were significantly higher than those from animals receiving dose-matched bolus injections after 2 and 4 weeks (p<0.05 and p<0.01, respectively). However, after boosting with a second bolus injection at week 4, titers were statistically similar between the formulations and their dose-matched bolus groups at weeks 6 and 8. Then at week 10, after the administration of the third bolus, animals receiving bolus BSA showed another spike in titers, resulting in significantly higher antibody titers compared to all dose-matched microsphere groups at that time point (p<0.001).

[0140] Although time-matched antibody titers would suggest the timing of antigen presentation, due to possible mismatches in the timing of antigen presentation (Paryani et al., J. Pediatr, 105:200-205 (1984); Barraclough et al., Am J Kidney Dis, 54:95-103 (2009)), peak antibody titers may be a more useful indicator of immune response in this case. Although the bolus injection schedule was chosen to time the microsphere burst in vitro, accelerated in vivo degradation may have led to offsetting antigen release. Consequently, at the end of the experiment, antibody titers in the microsphere group declined for several weeks, as would be expected in the absence of antigen, whereas titers in the bolus control group reached their highest levels after the third injection. Comparing peak antibody titers would help control for timing effects and determine which treatment is more immunogenic.

[0141] Antibody titers for formulations C, G, and E peaked at 13.9±1.3, 13.7±2.2, and 16.1±2.1 on the log2 scale, respectively, 4 weeks after microsphere administration, whereas the small and large dose-matched boluses peaked at log2 titers of 15.5±1.5 and 17.7±0.8, respectively, after 10 weeks (Figure 23). The Holm-Bonferroni correction method (Reverberi, Blood, 2002) recommended for multigroup titer comparisons was used. Transfus. 6:37-45 (2008)), formulations C and G (Figure 23A) induced peak antibody titers that were not statistically different from a dose-matched bolus control consisting of three injections of 22 μg BSA (p * =0.0645 and p * =0.0543). Formulation E (Figure 23B) also induced peak titers that were not statistically different from the dose-matched bolus control consisting of three bolus injections (p * =0.0784) (Table 5).

[0142] Example 7: PLGA Micromolded Particles for Drug Delivery material and method The development of single drug particles involves three major steps: 1) shell microfabrication, 2) vaccine / drug loading, and 3) particle sealing.

[0143] Microfabrication of the shell may involve mask fabrication (lithography), fluoro-mold fabrication (UV core) and heat pressing of PLGA (120°C for 30 min). The drug loading step may be performed by a Biodot robot, loading the core with a volume of 1.5 nL of drug. Sealing is achieved by placing a PLGA cap onto the shell and then sealing the particle at 37°C for 5 min and allowing the acetone to evaporate for 5 min.

[0144] Schematics of the process are presented in Figures 12A-12D and 24A-24C. These microparticles may be fabricated as sealed stacks at 37°C for 5 minutes each.

[0145] result The x, y, and z dimensions of the micromolded particle shell are 450 x 450 x 300 μm with a 100 x 100 x 100 μm core (cap dimensions are 450 x 450 x 150 μm) or 200 x 200 x 150 μm with a 100 x 100 x 100 μm core. These dimensions allow the particles to be delivered using a 21 gauge needle (inner diameter 514 μm) or a 23 gauge needle (inner diameter 337 μm).

[0146] Example 8. Single PLA particles for deposition of drug / sugar solution material and method IPV in 0.5M trehalose and 0.5M sucrose solution was deposited onto the PLA particles and then dried at room temperature in a stem cell culture hood. The PLA particles may be 3D printed using a Makerbot printer.

[0147] result This method allows the drug or antigen in sugar solution to be deposited onto the substrate, so that the drug or antigen is stabilized within the sugar glass, and the single PLA particles may serve as a useful platform for drying and delivery of drugs and antigens. Studies of the stability of IPV antigens on PLA particles are presented in the examples below.

[0148] Example 9: Stability of IPV microspheres The process for IPV microsphere formation presented in Example 1 above is fraught with challenges regarding IPV stability during the manufacturing process. These challenges are presented in Table 6 along with a summary of findings to overcome the challenges.

[0149] Table 6. Summary of barriers to IPV stability, approaches taken to overcome barriers, and findings. [Table 6]

[0150] Example 10: Co-encapsulation of IPV with gelatin or mild surfactant and sonication increases IPV stability during emulsification material and method IPV was mixed with water and / or excipients in a 100:1 w / w ratio. The excipients used were gelatin, maltodextrin, pullulan, myristic acid, and Tween 80. IPV with excipients was then added to DCM, or PLGA / DCM. The mixture was vortexed at the highest setting for 10 seconds or sonicated at 25% amplitude for 10 seconds. ELISA buffer (1% BSA, 1% Triton-X100 in PBS) was added, the mixture was vortexed at high setting for 10 seconds, and the layers were separated by centrifugation. The aqueous layer was removed and tested by ELISA.

[0151] result Both gelatin and Tween 80 improved the stability of IPV types I, II and III (Figure 25).

[0152] Types I and III IPV were generally the most sensitive to injury, with best recovery following sonication for 30 seconds at 20% amplitude (Figure 26).

[0153] Example 11: Drying IPV and excipients using Genevac improves IPV recovery material and method IPV was concentrated 26-fold and diluted with pullulan, pullulan / BSA, or sorbitol / MSG / MgCl 2 IPV was mixed with excipients at a mass ratio of 100:1, 350:1, or 700:1. IPV with excipients was dried by lyophilization (overnight) or using a Genevac (1 h, 30-35°C). After resuspension of the dried IPV with excipients, recovery was tested by ELISA.

[0154] result In all paired groups of lyophilized (Lyo) and Genevac (Figure 27), Genevac showed higher recovery than lyophilized.

[0155] Example 12: Freeze-dried IPV exhibits long-term stability at 37°C material and method IPV was mixed with the excipients 10% sorbitol, 8.5% MSG, and 8.5% MgCl 2 and lyophilized in plastic tubes. The lyophilized IPV with excipients was stored in plastic tubes in bags with desiccant in a humid environment at 37° C. for up to 30 days. At various time points, the lyophilized IPV was resuspended and tested for recovery by ELISA.

[0156] result Lyophilized forms of IPV types I, II and III with excipients showed long-term stability when stored at 37°C (Figure 2).

[0157] Example 13: Change in pH of the release medium by PLGA particles PLGA degrades by bulk erosion, resulting in acid accumulation inside the microspheres. During in vitro storage, limited volumes of buffer can cause acid accumulation in the tube, which can lead to particle erosion and acid accumulation inside the microspheres. For in vivo applications, the buffer in the local environment is constantly replenished, and acid accumulation outside the particles is minimal. Since PLGA degrades by bulk erosion, acid accumulation inside the microspheres can be problematic. Therefore, several PLGA molecules were tested for changes in pH of the release medium over time when the buffer was changed every 2-3 days or every 7 days.

[0158] result The results in Figure 29 demonstrate that the PLGA molecule alone changes the pH of the release medium, making it more acidic over time. Changing the buffer every 2-3 days helps to mitigate this effect, but does not solve the problem.

[0159] Example 14. Excipients that buffer the acidic production of PLGA microspheres It was observed that type I IPV was relatively stable at pH 7.4 at 37° C., and types II and III IPV were stable at pH 6-8 at 37° C. Therefore, several excipients were tested for their buffering capacity to buffer the acidic production of PLGA microspheres. material and method Mg(OH) 2 , Al(OH) 3 Buffering agents such as myristic acid and glycerol were incorporated into the PLGA microspheres to minimize changes in pH of the release medium. 2 Al(OH) has little or no solubility in water, it remains distributed throughout the polymer matrix and can potentially buffer internal compartments. 3 is a known adjuvant and can increase immunogenicity.

[0160] result In the results in Figure 28, Mg(OH) 2 It is demonstrated that the compound can buffer the acidic by-products in the release medium and stabilize the pH of the release medium at about pH 7. Example 15. IPV Microspheres Incorporating Stabilizing Excipients

[0161] Based on the investigations of Examples 9-14, PLGA microspheres were made incorporating IPV and various stabilizing additives. These formulations are presented in Table 7 below and were made using PLGA502H. Other formulations using higher molecular weight polymers, such as PLGA503H, are also contemplated. [Table 7]

[0162] Example 16. IPV stability after drying on PLA allows the use of lower excipient:vaccine ratios

[0163] Trivalent IPV 26x on PLA was dried for 16 hours at room temperature and humidity to test the retention of D antigen. The excipients used were 10% sorbitol, 8.5% MSG, 8.5% MgCl 2 The excipient to vaccine ratios tested were 300:1, 100:1, 60:1, 30:1, or no excipient. The percent of D antigen retained was assayed by ELISA.

[0164] result Upon drying of the solution onto PLA, no significant drop in stability was observed when the ratio was reduced to 30:1 (Figure 1). This data demonstrated that concentrated excipient-free IPV can be dried onto PLA cubes without significant loss of antigenic activity.

[0165] Example 17. Other formulations of concentrated IPV mixed with excipients and dried on PLA cubes The formulations listed in Table 8 represent the % D antigen retained when concentrated IPV was mixed with the indicated excipient solutions ("Polyol" and "Other" columns) in the indicated ratios ("Excipient: Vaccine" column, all indicated ratios are 100:1) and allowed to dry overnight on PLA cubes.

[0166] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the disclosed invention belongs. The literature and materials cited herein are specifically incorporated by reference. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is intended that such equivalents be encompassed by the following claims. [Table 8]

Claims

1. 1. A plurality of polymeric microparticles comprising a biodegradable and biocompatible polymeric shell defining a core space containing an agent selected from the group consisting of therapeutic, prophylactic or diagnostic agents, the shell being a multi-layered structure and produced by three-dimensional printing or micromolding, the agent being released from the core space of the biocompatible polymeric shell upon degradation, the plurality of polymeric microparticles being suitable for administration to an individual.

2. 13. The plurality of polymeric microparticles of claim 1, fabricated by three-dimensional printing.

3. 3. The plurality of polymeric microparticles of claim 1 or 2, wherein the agent is filled, pipetted or jetted into the core space.

4. The plurality of polymeric microparticles according to any one of claims 1 to 3, wherein the core space is capped.

5. 5. The plurality of polymeric microparticles of any of claims 1 to 4, which are deliverable through a 21 gauge needle.

6. 5. The plurality of polymeric microparticles of any of claims 1 to 4, which are deliverable through a 23 gauge needle.

7. 7. The plurality of polymeric microparticles of any one of claims 1 to 6, wherein the plurality of polymeric microparticles comprises a stabilizing agent, the stabilizing agent comprising a compound selected from the group consisting of sugars, oils, lipids and carbohydrates, preferably a sugar, most preferably a sugar selected from the group consisting of sucrose, trehalose and combinations thereof, optionally in the form of a sugar glass.

8. 8. The plurality of polymeric microparticles of any one of claims 1 to 7, wherein the polymer is hydrolytically biodegradable, preferably a polyester, and most preferably selected from the group consisting of poly(lactic acid), poly(glycolic acid), and copolymers thereof.

9. A plurality of polymeric microparticles according to any one of claims 1 to 8, providing release at intervals of 10 to 90 days, preferably at intervals of 30 to 60 days.

10. 10. The plurality of polymeric microparticles of any one of claims 1 to 9, wherein the plurality of polymeric microparticles is injectable or implantable or applicable to a mucosal surface selected from the group consisting of nasal, pulmonary, oral, vaginal and rectal surfaces.

11. 11. The plurality of polymer microparticles of any one of claims 1 to 10, wherein the plurality of polymer microparticles comprises the polymer that is poly(lactic-co-glycolic acid) (PLGA), and optionally the plurality of polymer microparticles further comprises a buffering agent, and optionally the buffering agent is selected from the group consisting of magnesium hydroxide, aluminum hydroxide, and myristic acid.

12. 13. The plurality of polymeric microparticles of claim 1 formed by micromolding, the plurality of polymeric microparticles comprising uniform micrometer-scale dimensions and drug loading within a population of the plurality of polymeric microparticles.

13. A plurality of polymeric microparticles according to any one of claims 1 to 12 for use in a method of delivering a medicament to an individual in need thereof.

Images