Composition for targeted delivery of therapeutic compounds to bone tissue

Abstract

A system and method are disclosed for the production of drug delivery vesicles formed from hydroxyapatite (Ca10(PO4)6(OH)2), called apasomes, using a microfluidic device. The device comprises a first inlet with a bifurcated tube and a second inlet with an inlet tube. Hydroxyapatite nanoparticles and, optionally, therapeutic agents suspended in a second fluid are introduced into the device via the second inlet, while a miscible first fluid is introduced via the first inlet. The two fluids are combined at a mixing point within the device and the combination fluid travels through an elongate tube before exiting the device. Shear forces cause self-assembly of vesicles, the size of which can be modified by adjusting the flow rates, the shapes and dimensions of the tube, and the composition of the fluids. The method may also include refluxing, filtration and drying steps to enable scalable production of customizable drug delivery vesicles for therapeutic applications.

Description

COMPOSITION FOR THE TARGETED ADMINISTRATION OF THERAPEUTIC COMPOUNDS TO BONE TISSUE CROSS REFERENCE TO A RELATED APPLICATION

[0001] This application claims priority over United States Provisional Application No. 63 / 666,085, filed on June 28, 2024, which is a U.S. provisional application, the contents of which are expressly incorporated herein by reference in their entirety. FIELD OF DISCLOSURE

[0002] This disclosure relates to a pharmaceutical composition for use in drug delivery. Specifically, this disclosure covers the chemical structure of a drug delivery composition that can be used to facilitate targeted drug delivery to bone tissue, along with its application in pharmaceutical formulations for oral and / or topical administration. INTRODUCTION

[0003] Bone-related diseases present significant therapeutic challenges, requiring the development of innovative therapeutic approaches that address the stark contrast between typical organ-compatible treatments and treatments designed to be chemically compatible with bone tissue.

[0004] According to the International Osteoporosis Foundation (IOF), an estimated 200 million people worldwide suffer from osteoporosis. Each year, osteoporosis causes more than 8.9 million fractures globally, resulting in over $20 billion in healthcare costs in the United States alone. Other bone conditions, such as osteoarthritis, bone cancer, and osteomyelitis, have been on the rise in recent years and are projected to become even more prevalent in the near future. By 2040, it is estimated that more than 78 million adults in the United States will be diagnosed with osteoarthritis, nearly double the 32.5 million adults currently affected by these conditions.

[0005] In recent decades, numerous drugs have been developed to treat bone diseases. These medications have proven effective in treating bone conditions; however, they can cause significant side effects in patients when released outside of bone tissue. This is especially problematic in the case of potent and powerful therapeutic remedies, such as treatments for bone cancer.

[0006] To minimize side effects and discomfort experienced by patients receiving treatment for bone conditions, it would be desirable to use a drug delivery system that limits the release of therapeutic agents solely to bone tissue. However, the chemistry of the bone microenvironment presents a significant challenge for drug delivery, as the structure and physiology of this tissue are drastically different from those of other tissues. Effective drug delivery for the treatment of bone conditions requires a vehicle capable of overcoming unique barriers of bone tissue, such as poor vascularization, dense mineralized matrix, and limited permeability, in order to deliver therapeutic treatments to the desired areas of the tissue.

[0007] Therefore, it would be desirable to use drug delivery vehicles that can take the form of nanohydroxyapatite (nHA) nanoparticles formed using microfluidics, to enable targeted delivery and release of therapeutic agents into bone tissue. The proposed system takes advantage of the fact that nHA is a naturally occurring mineral component of bone, allowing for the controlled release of therapeutic agents within bone tissue and limiting their release outside of it. SUMMARY

[0008] In some respects, this disclosure refers to a vehicle for the delivery of therapeutic agents to bone tissue, where the vehicle is an "apasome." As used in this disclosure, the term apasome may refer to nanometric or micrometric bodies formed from a compound having the following formula: Cal0(PO4)6(OH)2 (“nHA” or “hydroxyapatite”). The apasomes of this disclosure may be hydroxyapatite nanoparticles that self-assemble to form a vesicle or vacuole. Apasomes may be designed to encapsulate, transport, and release therapeutic agents in specific environments. For example, apasomes formed from nHA may be used to deliver therapeutic agents to bone tissue.

[0009] The use of nHA offers opportunities to develop new medical treatments and drug delivery methods that require lower doses of medication and allow users to avoid many of the side effects that would accompany the widespread delivery of such drugs to all types of tissue, by improving targeted delivery to the patient's bones.

[0010] In one embodiment of this disclosure, an apasome may be formed from nHA encapsulating any predetermined therapeutic compound. The therapeutic compounds encapsulated by the nHA apasome may include any therapeutic agent known in the art.

[0011] This disclosure may include a device and method for fabricating apasomes from nHA. The microfluidic device may include a first inlet, a second inlet, and an outlet. The first inlet may be connected to a first reservoir and allow the delivery of a first fluid to a mixing point via a branch tube. Each end of the branch tube may have a stirring length and an outlet. The branch tube may be fluidly coupled to the first inlet via an inlet stirring tube. The second inlet and the first inlet may be fluidly coupled at a mixing point, where the first and second fluids can mix to form a third fluid. The third fluid may pass through a flow focusing tube, where shear forces (i.e., mixing) can promote apasome formation before the third fluid exits the microfluidic device.The flow focusing tube can be a straight cylindrical tube.

[0012] The second fluid can be a nanoparticle suspension containing nHA nanoparticles, a fatty acid, a solvent, and optionally, a therapeutic agent or marker. The first fluid can be a solvent miscible with the second fluid.

[0013] This disclosure also claims a method for producing apasomas by combining a suspension of nHA nanoparticles, which also contains a fatty acid, a solvent, and an optional therapeutic agent, with another solution. The method comprises the steps of introducing a first and second fluid into the first and second inlets of a microfluidic device, respectively. The method may also comprise moving the first and second fluids through the microfluidic device by means of a pressure-induced flow and combining them at a mixing point. to form a third fluid. The method may include passing the third fluid through a flow focusing tube (in some embodiments, a straight cylindrical tube), which may promote apasome self-assembly. The apasome-containing third fluid may be collected from the microfluidic device or recirculated by reflux through the device via the first and / or second inlet.

[0014] The resulting apasomes can have a diameter between 50 nm and 200 nm. The size of the apasomes can be adjusted by changing the flow rate of the first solution, the flow rate of the second solution, the dimensions of the microfluidic device components, including the diameter, shape, and length of the tube, as well as by changing the fatty acid composition in the second fluid.

[0015] The second fluid can be prepared by combining nHA with a solvent and a fatty acid selected from the following: octanoic acid, nonanoic acid, decanoic acid, lauric acid, stearic acid, and caprylic acid. The second fluid can contain any fatty acid with an aliphatic chain of between 8 and 18 carbon atoms. The second fluid can also contain suspended nanoparticles that have been treated with a therapeutic agent before the second fluid is introduced into the microfluidic device.

[0016] The microfluidic device may also include a filter configured to separate particles larger than 400 nm in diameter from other apasoms. In one embodiment, the microfluidic device includes a reflux loop that recirculates the fluid through the device, from the outlet to the first and / or second inlet. In another embodiment, the fluid is pumped through the microfluidic device.

[0017] This disclosure also teaches a method for forming nHA apasomas, in which a multi-inlet device can be used to combine water with an nHA-containing suspension. The nHA-containing suspension can be a colloidal suspension of nHA in isopropanol. The nHA-containing suspension can be pretreated with a metal or other ion, or with a therapeutic agent.

[0018] Additional aspects related to this disclosure are set out, in part, in the description that follows, and in part will be evident from it, or can be learned through the practice of this disclosure.

[0019] It is understood that both the above and the following descriptions are illustrative and explanatory only, and are not intended to limit the claimed disclosure or its application in any way. DESCRIPTION OF THE DRAWINGS

[0020] The incorporated drawings, which form part of this specification, exemplify aspects of this disclosure and, together with the description, explain and illustrate its principles. Any measurements that may be included as part of any accompanying or incorporated drawing are intended to describe one possible embodiment of this disclosure and should not be construed as limiting the actual or relative size of any part or component thereof. Empirical data or numerical ranges described herein are cited for illustrative purposes, unless otherwise stated, to demonstrate the application of the systems and methods described throughout this document.

[0021] FIG. 1 is a perspective view of a modality of a microdevice with at least one input.

[0022] FIG. 2 is a partial perspective view of a modality of a microdevice with at least one input.

[0023] FIG. 3 is a detailed perspective view of the combination point of a microdevice with at least one input and of the surrounding components.

[0024] FIG. 4 illustrates an example of the effect that altering a microfluidic assembly parameter can have on the size of the resulting apasoms.

[0025] FIG. 5 illustrates an example of the effect that changing the fatty acid (FA) identity can have on the size of the resulting apasomas.

[0026] FIG. 6 is a high-level process diagram illustrating the steps of the method in this disclosure.

[0027] FIG. 7 is an electron microscopy image of one modality of the present disclosure.

[0028] FIG. 8 is a photomicrograph of a fluorescent modality of the present disclosure that contains europium.

[0029] FIG. 9 contains the result of the thermogravimetric analysis of one modality of the present disclosure.

[0030] FIG. 10 contains the infrared spectroscopy result of one modality of the present disclosure.

[0031] FIG. 11 contains a fluorescent micrograph measuring the fluorescent activity of one modality of the present disclosure.

[0032] FIG. 12 contains the result of the thermogravimetric analysis of one modality of the present disclosure with ibuprofen chemically absorbed by the second solution.

[0033] FIG. 13 contains the infrared spectroscopy result of one modality of the present disclosure.

[0034] FIG. 14 contains the resulting distribution of apasome size measured by dynamic light scattering (DLS) testing.

[0035] FIG. 15 contains the results of a cell viability assay testing various modalities of the present disclosure. DETAILED DESCRIPTION

[0036] The following detailed description describes drug delivery nanoparticles and chemical compositions and may refer to one or more figures. These figures should not be interpreted in a limiting sense and are included only as illustrative examples of possible embodiments of the compound in this disclosure. The figures depict the structure of the composition of this disclosure in sufficient detail to enable persons skilled in the art to practice the invention. It should be understood that other implementations or devices may be used, and that structural changes and / or substitutions of various functional groups may be made without departing from the scope and spirit of this disclosure.

[0037] The drug delivery vesicles described in this disclosure may be useful in the treatment of bone conditions such as degenerative bone diseases and cancers. They can be administered orally, intravenously, and / or transdermally. Drug delivery vesicles can be used to transport therapeutic agents, such as drugs or genetic material, to specific targets in the body. These vesicles may include various structures such as polymeric nanoparticles, micelles, and liposomes, among others, and can be customized according to the properties of the drug to be delivered and the target tissue. This disclosure may refer to an “apasome.”

[0038] The apasomes described herein may be formed from nanohydroxyapatite (nHA), which may have the following structure: Cait^PChXOH)?. The apasomes described herein may be designed, configured, or formulated to enable targeted drug delivery to bone tissue. It may be desirable to use therapeutic agents that have an affinity for nHA (e.g., ibuprofen, doxorubicin, and imaging agents such as europium (Eu) or Nile Red (NR)). However, it is envisaged that the apasomes may be useful for delivering any therapeutic agent or drug known in the art.

[0039] Apasomes can be assembled using a microdevice with at least two inlets. A purely illustrative example of a microfluidic assembly is shown in FIG. 1. In one embodiment, the microfluidic assembly 100 can have one or more inlets. In a particularly suitable embodiment, the microfluidic assembly 100 can have a water inlet 105 and a suspension inlet 150. The water inlet 105 can consist of a water reservoir 110 and a bifurcated water inlet tube 115. The water inlet reservoir 110 can have a sidewall 112. The sidewall 112 can be arranged or configured to have a conical shape, the narrower end of which can be closest to the bifurcated water inlet tube 115. The sidewall 112 can be smooth or textured according to user preferences and the inlet fluid conditions. By way of non-limiting example, in one embodiment, the The side wall 112 may have one or more inwardly extending protrusions that can serve to agitate the inlet fluid, promoting non-laminar flow and thus homogenizing the fluid. The bifurcated water inlet tube 115 may have a first outlet 130 and a second outlet 135. This tube may be coiled, bent, or twisted along a first agitation region 120 and a second agitation region 125 to promote homogeneity of the fluid passing through it.

[0040] Referring to FIG. 2, the first outlet 130 and the second outlet 135 can converge at a junction point 140. Both outlets can be fluidly coupled to the water reservoir 110 via the bifurcated tube. As shown in FIG. 1, these outlets can be positioned at a predetermined distance below the water reservoir 110. The relative position of the outlets with respect to the reservoir can control the head pressure difference that determines the fluid flow velocity. In a particularly desirable embodiment, the first and second outlets can be approximately 6.976 pm below the water reservoir 110. This distance can allow for a particularly suitable flow velocity when the vertical distance between the upper 200 and lower 210 of the microfluidic assembly is determined. Alternatively, a pump can control the fluid flow.

[0041] As shown in FIG. 2, the assembly may also include a suspension inlet 150. This may include a reservoir 155 and a suspension inlet tube 160. The reservoir 155 may have a conical side wall 158, with its narrower end closest to the tube 160. The wall 158 may be smooth or textured depending on the fluid conditions and user preferences. By way of non-limiting example, this wall may have one or more internal protrusions that agitate the suspension fluid, promoting non-laminar flow and homogenizing the fluid. In an embodiment where the suspension is a mixture of nHA and isopropanol, the wall may have protrusions arranged as "islands." The fluid velocity may be controlled by a pressure differential generated by gravity or by a pump.

[0042] The tube 160 can be seamlessly coupled to the reservoir 155 at one end and to the combining point 140 at the other. By way of non-limiting example, the tube can have a diameter that allows the passage of a solution of isopropanol and hydroxyapatite nanoparticles. It can also be bent, coiled, or configured to promote fluid agitation over an agitation length 165. This prevents sedimentation of the particles and promotes a homogeneous concentration of nHA apasomes. After agitation, the suspension solution can exit through a third outlet 170.

[0043] In FIG. 3, the combining point 140 can be seamlessly coupled to outlets 130, 135, and 170. By way of non-limiting example, outlets 130 and 135 can supply water at a predetermined flow rate, and at the combining point, this water will mix with the suspension in tube 160 to form a fluidized product of apasomas. This fluid will then pass through a flow focusing tube 175 to outlet 180. The tube 175 can be of a suitable width to allow laminar flow, promoting uniform dilution of the suspension. In a preferred embodiment, the diameter of the tube can be between 50 µm and 200 µm, preferably 100 µm.

[0044] Since the fluid can flow in laminar flow, the parabolic velocity profile can encourage the exchange of fluid layers, promoting uniform mixing. The product fluid can exit through outlet 180 and be stored in any suitable container, ideally under refrigeration (278K) to prevent denaturation.

[0045] Furthermore, the microfluidic assembly can be arranged so that a pressure difference moves the fluids. This difference can be generated by the height difference between the inlet and outlet (for example, 78 mm) or by a pump. This eliminates the need for electric pumps and ensures consistent process parameters.

[0046] The product fluid may contain self-assembled apasoms. The physical parameters of the device (height, flow rate, diameter) can be adjusted to vary the size of the apasoms, which can range from 170 nm to 400 nm, depending on the therapeutic agent to be encapsulated.

[0047] FIG. 4 is presented as a non-limiting example of the effect of modifying a parameter on the size of the apasome.

[0048] To produce apasomas, the suspension fluid can be a colloidal suspension of nHA nanoparticles in isopropanol. The process begins with pure nHA dissolved in a solvent with modifying reagents. Prior to this, a first solution of a fatty acid (5% to 15%) in anhydrous acetone is prepared, preferably 10% lauric acid. The acid can be saturated with 8 to 12 carbon atoms. Examples include octanoic, nonanoic, decanoic, lauric, stearic, and caprylic acids.

[0049] The fatty acid influences the size of the apasome. As shown in Figure 5, the longer the fatty acid chain, the larger the apasome, possibly due to more rigid packing. This also affects the release of the agent: short acids like caprylic acid generate small apasomes for rapid release; long acids like stearic acid generate large apasomes for sustained release.

[0050] Pure nHA is added to the first solution to obtain a second one. For apasoms of 170-400nm, the particle size should be between 20nm and 50nm, preferably 20nm.

[0051] The final nHA concentration can be between 50 and 100 mg / mL; an example is 75 mg / mL. The second solution can be refluxed to homogenize the particles, break up agglomerates, and modify their polarity (slight hydrophobicity), facilitating self-assembly. The particles are then washed with acetone and dried (e.g., at 373 K). A high-frequency sonicator can be used to prepare the isopropanol suspension, which is filtered (e.g., removing particles >400 nm).

[0052] Other parameters that affect the size and properties of the apasome are the inflow velocities. Higher velocities generate greater shear, faster mixing, and smaller vesicles, as shown in FIG. 4. It also affects the efficiency of drug encapsulation.

[0053] In addition to velocity, the ratio of inflow rates affects size. A 5:1 ratio (water:suspension) produces smaller, more uniform apasomas than a 1:1 ratio, due to faster dilution. Figure 7 shows size differences in similar samples analyzed by TEM.

[0054] Apasomes can be optimized according to their use. For intravenous formulations, smaller apasomes (>200 nm) are preferred due to their bioavailability. For sustained release or high-load applications, larger apasomes are preferred. However, these can be detected by the immune system. An apasome can contain more than one therapeutic agent.

[0055] Apasomas can have a spherical and multilayered structure. The outer layer of nHA may be substantially non-porous. The interior may be empty or contain water, a solution, or a therapeutic agent.

[0056] In another aspect, the composition can be combined with others to enhance intravenous, transdermal, or oral treatments. For example, for oral use, it may contain inactive excipients; for transdermal use, active excipients that improve the therapeutic effect.

[0057] Apasomes can contain integrated or adsorbed metal ions, such as magnesium, strontium, europium, barium, etc. For example, a metallic salt solution (CuSCh, FeCl, ZnCl) can be added to the second fluid to form charged nanoparticles. These can confer osteogenic, luminescent, or antimicrobial properties. Alternatively, the metal salt can be introduced directly into the device. The ion can then be embedded in the core, surface, or lipid structure. For example, europium-containing apasomes exhibit fluorescence (FIG. 8).

[0058] Other compounds such as carbonates, fluorides or other ions can also be incorporated, modifying the release (controlled by pH) or bone affinity, as well as the solubility or biochemical effect.

[0059] Referring to FIG. 6, the disclosure includes a method 900 for producing apasomas. Step 905: Prepare first fluid. Step 915: Prepare second fluid (nHA + solvent + fatty acid, optionally therapeutic agent). Step 910: Introduce first fluid at a specific flow rate into a bifurcated inlet with a reservoir. Step 920: Introduce second fluid. Step 930: Mix at a combining point to form a third fluid. Step 940: Agitate this fluid in a focusing tube. Step 950: Discharge the fluid, or alternatively, reflux in step 970.

[0060] Fluids can flow at any suitable flow rate. For example, between 0.5 and 40 pL / min, and solutions between 0.25 pL / min and 20 pL / min.

[0061] Furthermore, the composition may have other, as yet unexplored, medical benefits. Preliminary results indicate efficacy in encapsulating mRNA or other genetic material. It is envisioned that it could be applied to other therapies through similar mechanisms. Applications for delivering antibacterial, antimicrobial, antiviral, or anticancer treatments are also anticipated.

[0062] The following examples illustrate the invention and should not be interpreted as limiting. The details are presented as non-exhaustive examples for the purpose of illustrative discussion of certain embodiments of the invention.

[0063] Example 1 - Nile Red (NR) Chemoabsorption

[0064] To evaluate the ability of nHA to chemically absorb Nile Red, pure nHA was first dissolved in water, and NR was added to the nHA-water solution at concentrations ranging from 0.1% to 5%. The NR+nHA solution was stirred for 24–72 hours on a reciprocating shaker, then centrifuged and washed to remove any unbound or excess NR. As illustrated in Figure 9, which shows the thermogravimetric analysis (TGA) results, NR was chemically bound to the nHA during this process. As shown in Figure 10, the infrared (IR) absorption spectrum of the NR-treated nHA exhibited distinct peaks between 2000 and 1500 cm⁻¹, further confirming that NR was chemoabsorbed by the nHA. Following this test, the NR-treated nHA was used to form apasomas using the processes described above. An exemplary image of the resulting NR-treated apasomas is shown in FIG. 11.The distinctive fluorescence of the apasomes captured in this image confirms that NR was successfully incorporated into the apasomes.

[0065] Example 2 - Ibuprofen Chemoabsorption

[0066] Tests with ibuprofen-treated nHA can be performed using a method similar to that in Example 1. To confirm the chemical uptake of ibuprofen by the nHA, ibuprofen and nHA were weighed in different ratios, all between 4:1 and 2:1. The ibuprofen and nHA were dissolved in pure water and stirred on a reciprocating shaker for 24–72 hours. After stirring, the ibuprofen-nHA solution was centrifuged and washed with pure water to remove excess or unbound ibuprofen. Figures 12–14 show the TGA, IR, and dynamic light scattering (DLS) results for the ibuprofen-treated nHA. Mass loss around 150 °C indicates the presence of ibuprofen in the nHA. The presence of IR bands between 300 and 1500 cm⁻¹ is also indicative of the reaction of ibuprofen with nHA. Finally, the ibuprofen-treated nHA was used to produce apasomas using the microfluidic device described here, as shown in the figures.12 and 13, the size of the apasomas treated with ibuprofen was approximately equal to the size of the apasomas not treated with ibuprofen.

[0067] Example 3 - In Vitro Test

[0068] To evaluate the cytotoxicity of nHA apasomes produced according to this disclosure, in vitro tests were performed using HeLa cells. HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with penicillin-streptomycin. The cells were maintained in a humidified incubator at 37°C and seeded at a density of 100 cells / pL in a 96-well plate, incubated for 24 hours to allow cell adhesion. After cell adhesion, the apasoms were introduced into the cells in triplicate. Control wells received an equal volume of apasom-free solution.

[0069] After 24 hours of incubation, cell viability was assessed using an MTS assay. The test steps included adding MTS reagent to each plate, incubating the samples for 20 minutes, and measuring the absorbance at a wavelength of 490 nm using a microplate reader. As illustrated in Figure 15, treatment with apasomes, including those containing therapeutic reagents, resulted in minimal reduction in cell viability (>90% viability maintained). These results indicate that nHA apasomes formed by microfluidic assembly are well tolerated by HeLa cells. The low cytotoxicity, along with the results of the aforementioned chemoabsorption examples, supports the use of nHA apasomes for drug delivery.

[0070] To further evaluate the cellular internalization of apasomas, fluorescence microscopy was performed using an EVOS m5000 microscope. A red fluorescence filter (i.e., emission wavelength range -580-620 nm) was used to detect apasomas labeled with Nile Red. As shown in Figure 11, which contains fluorescent images of one embodiment of the present disclosure, apasomas labeled with Nile Red can be identified by their fluorescent properties.

[0071] Although specific configurations are disclosed here, it is envisaged that other configurations with different formations and structures may be achievable. It is also envisaged that slight variations to the configurations mentioned may be implemented to obtain similar results, and such configurations are considered within the scope of this disclosure.

[0072] Finally, other implementations of the disclosure will become evident to those skilled in the art upon consideration of the specification and practice of the disclosure herein revealed. The specification and examples are intended to be considered illustrative only, and the true scope and spirit of the disclosure are indicated by the following claims.

[0073] This disclosure provides the following non-limiting items: a system for manufacturing drug delivery vesicles from Calcium(PO4)6(OH)2 using a microfluidic device, said microfluidic device comprising a first inlet, with a first reservoir and a bifurcated tube. The bifurcated tube may have a left portion and a right portion. The left portion may comprise a left stirring length and a left outlet, and the right portion may comprise a right stirring length and a right outlet. The microfluidic device may also comprise a second inlet, including a second reservoir and a secondary inlet tube. The secondary inlet tube may comprise a secondary stirring length and a secondary outlet, wherein the secondary inlet tube is fluidly coupled to the second reservoir and the secondary outlet.The outlet of the secondary tube may be positioned below the second reservoir. The microfluidic device may also include a flow focusing tube fluidly coupled to the outlet of the secondary tube, the right outlet, and the left outlet. The flow focusing tube may be a straight cylindrical tube, and the fluids from the first and second reservoirs may mix within the flow focusing tube. The microfluidic device may also include an outlet fluidly coupled to the flow focusing tube.

[0074] This disclosure also provides a system for fabricating drug delivery vesicles, wherein the drug delivery vesicles are apasoms (defined herein) and are formed by self-assembly in the microfluidic device. This disclosure further provides a system for fabricating drug delivery vesicles wherein a therapeutic drug is combined with a suspension of Cal0(PO4)6(OH)2 nanoparticles to form a therapeutic nanoparticle suspension, and such suspension is introduced into the microfluidic device through the second inlet.

[0075] This disclosure also provides the following non-limiting item: a method for manufacturing drug delivery vesicles from CalO(PO4)6(OH)2 by means of a microfluidic device, wherein: a first fluid is introduced into the microfluidic device through a first inlet at a first inlet flow rate, said first inlet comprising a first reservoir and a bifurcated tube with a left portion and a right portion. For this particular item, the left portion comprises a left stirring length and a left outlet, and the right portion comprises a right stirring length and a right outlet. In this item, a second fluid is introduced into the microfluidic device through a second inlet at a second inlet flow rate, said second inlet comprising a second reservoir and a secondary inlet tube. The secondary inlet tube may comprise a secondary tube stirring length and a secondary tube outlet, and may be fluidly coupled to the second reservoir and the secondary tube outlet.For this item, the outlet of the secondary tube can be positioned below the second reservoir. The method can also include the step of forming a third fluid by combining the first and second fluids in a flow focusing tube fluidly coupled to the outlet of the secondary tube, the right outlet, and the left outlet. For this method, the flow focusing tube is a straight cylindrical tube, and the third fluid is mixed within the flow focusing tube. For this method, the third fluid can exit the microfluidic device through an outlet, where the outlet is fluidly coupled to the flow focusing tube.

[0076] Drug delivery vesicles fabricated by this method can be apasoms (defined here) formed by self-assembly in the microfluidic device. In a non-limiting example of the method, the first fluid is water, the second fluid is a combination of a therapeutic drug and suspended Cal0(PO4)6(OH)2 nanoparticles, and the third fluid is a suspension of drug delivery vesicles containing the therapeutic drug.

[0077] Drug delivery vesicles can have a diameter between 50 nm and 200 nm. The size of the drug delivery vesicles can be adjusted by altering one or more of the following: a. the flow rate of the first inlet; b. the flow rate of the second inlet; c. the diameter of the flow focusing tube; d. the vertical difference between the first inlet, the second inlet, and the outlet; and e. the composition of the second fluid.

[0078] For this method, suspended CalO(PO4)6(OH)2 nanoparticles can be prepared by combining pure CalO(PO4)6(OH)2 with a solvent and a fatty acid selected from: octanoic acid, nonanoic acid, decanoic acid, lauric acid, stearic acid, and caprylic acid. In one embodiment of this method, suspended CalO(PO4)6(OH)2 nanoparticles can be prepared by combining pure CalO(PO4)6(OH)2 with a solvent and any fatty acid containing between 8 and 18 carbon atoms in its aliphatic chain. The method may also include the step of treating the suspended CalO(PO4)6(OH)2 nanoparticles with a therapeutic agent before introducing the second fluid into the microfluidic device. The microfluidic device may further comprise a filter configured to separate particles with a diameter greater than 400 nm from the apasomes. The microfluidic device may be operated by a pressure differential generated by a pump.The method may also include the steps of refluxing the second fluid before introducing it into the microfluidic device and / or filtering the third fluid to separate particles with a diameter greater than 400 nm.

Claims

CLAIMS 1. System for manufacturing drug-releasing vesicles from Cait^PChXOH)? using a microfluidic device, the microfluidic device comprising: a first inlet conduit, further comprising a first reservoir and a bifurcated tube, wherein the bifurcated tube has a left portion and a right portion, wherein the left portion comprises a left stirring length and a left outlet, and wherein the right portion comprises a right stirring length and a right outlet, a second inlet conduit, further comprising a second reservoir and a second inlet tube, wherein the second inlet tube comprises a second tube stirring length and a second tube outlet, wherein the second inlet tube is fluidly coupled to the second reservoir and the second tube outlet, and wherein the second tube outlet is below the second reservoir,a flow focusing tube fluidly coupled to the outlet of the second tube, the right outlet and the left outlet, wherein the flow focusing tube is a straight cylindrical tube, and wherein the fluids from the first reservoir and the second reservoir are mixed in the flow focusing tube, and an outlet fluidly coupled to the flow focusing tube.

2. The system of claim 1, wherein the drug-releasing vesicles are apasoms formed by self-assembly in the microfluidic device.

3. The system of claim 1, wherein a therapeutic drug is combined with a suspension of Caio(PO4)e(OH)2 nanoparticles to form a therapeutic nanoparticle suspension, and the therapeutic nanoparticle suspension is introduced into the microfluidic device through the second inlet conduit.

4. A method for manufacturing drug-releasing vesicles from Cait^PChXOH)? using a microfluidic device, wherein: a first fluid is introduced into the microfluidic device through a first inlet conduit at a first inlet flow rate, the first inlet conduit further comprising a first reservoir and a bifurcated tube, wherein the bifurcated tube has a left portion and a right portion, wherein the left portion comprises a left stirring length and a left outlet, and wherein the right portion comprises a right stirring length and a right outlet; a second fluid is introduced into the microfluidic device through a second inlet conduit at a second inlet flow rate, the second inlet conduit further comprising a second reservoir and a second inlet tube.wherein the second inlet tube comprises a second tube stirring length and a second tube outlet, wherein the second inlet tube is fluidly coupled to the second reservoir and the outlet of the second tube, and wherein the outlet of the second tube is below the second reservoir, a third fluid is formed by combining the first fluid and the second fluid in a flow focusing tube fluidly coupled to the outlet of the second tube, the right outlet and the left outlet, wherein the flow focusing tube is a straight cylindrical tube, and wherein the third fluid is mixed in the flow focusing tube, and the third fluid exits the microfluidic device through an outlet, wherein the outlet is fluidly coupled to the flow focusing tube.

5. The method of claim 4, wherein the drug-releasing vesicles are apasoms formed by self-assembly in the microfluidic device.

6. The method of claim 4, wherein the first fluid is water, the second fluid is a combination of a therapeutic drug and CaXPChXOH nanoparticles in suspension, and the third fluid is a suspension of drug-releasing vesicles containing the therapeutic drug.

7. The method of claim 4, wherein the drug-releasing vesicles have a diameter between 50nm and 200nm.

8. The method of claim 6, wherein the size of the drug-releasing vesicles is adjusted by modifying one or more of the following parameters: a. the inlet flow rate of the first conduit; b. the inlet flow rate of the second conduit; c. the diameter of the flow focusing tube; d. the vertical difference between the first inlet conduit, the second inlet conduit, and the outlet; and e. the composition of the second fluid.

9. The method of claim 6, wherein the suspended CaXPChXOH nanoparticles are prepared by combining pure CaXPChXOH with a solvent and a fatty acid selected from: octanoic acid, nonanoic acid, decanoic acid, lauric acid, stearic acid, caprylic acid.

10. The method of claim 6, wherein the CaXPChXOH nanoparticles in suspension are prepared by combining pure CaXPChXOH with a solvent and a fatty acid containing between 8 and 18 carbon atoms in its aliphatic chain.

11. The method of claim 4, wherein the CaXPChXOH nanoparticles in suspension are treated with a therapeutic agent before the second fluid is introduced into the microfluidic device.

12. The method of claim 4, wherein the microfluidic device includes a filter configured to separate particles larger than 400nm in diameter from the apasomes.

13. The method of claim 4, wherein the microfluidic device operates by means of a pressure differential generated by a pump.

14. The method of claim 4, further comprising the reflux of the second fluid before being introduced into the microfluidic device.

15. The method of claim 4, further comprising filtration of the third fluid to separate particles larger than 400nm in diameter.

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