Microparticle formulations for intravenous therapy and methods of making and using same

Abstract

The present disclosure provides methods and formulations for therapeutic microparticles, which are used to administer the therapeutic microparticles to a subject via intravenous injection. It also provides methods for administering the therapeutic microparticles to a subject in need thereof. The formulations remain stable for up to three hours and prevent precipitation and aggregation of the therapeutic microparticles in solution.

Description

[Technical Field]

[0001] government support This invention was made with government support under Grant Nos. W81XWH-12-1-0414 and W81XWH-17-1-0389 awarded by the Department of Defense. The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims priority to U.S. Provisional Patent Application No. 63 / 409,935, filed September 26, 2022, the entire contents of which are incorporated by reference.

[0003] The present disclosure relates generally to the field of pharmacology. In particular, the present disclosure provides a formulation suspending therapeutic microparticles, as well as methods for making and using the same. The formulation allows the therapeutic microparticles to be administered to a subject via intravenous infusion. The present disclosure also provides a method for administering the therapeutic microparticles to a subject in need thereof. [Background technology]

[0004] Intravenous therapy is a medical technique that administers fluids, medications, and nutrients directly into a patient's circulatory system. Intravenous therapy is one of the most common and medically accepted systems for administering active agents or drugs to patients. This method of administering drugs and fluids is useful in situations where medications must be administered to an unconscious or unable to swallow individual. Intravenous therapy is considered the most rapid method of administering medications to a patient. Intravenous therapy also allows physicians to control the rate at which active ingredients are administered over time simply by controlling the rate at which the intravenous (IV) infusion enters the patient's circulatory system. There are also numerous therapeutic agents, particularly in the field of nanomedicine, for which intravenous (IV) therapy is the only or preferred method for administering a given therapeutic agent.

[0005] Because intravenous therapy administers an active agent directly into a subject's circulatory system, a major challenge with intravenous therapy is ensuring that the active agent does not precipitate or form clumps before, during, or after injection into a patient's veins and arteries. Even minimal clumping within a patient's veins can lead to blood clots, arterial blockages, stroke, and heart failure. In practice, most IV preparations are typically compounded by a pharmacist before being scheduled for administration intravenously to a patient. Given the actual operating conditions in a hospital or pharmacy, as well as the standard range of IV infusion rates, the active agent must remain suspended for at least 30 minutes.

[0006] There is a need for a formulation suitable for intravenous administration to the patient's body, one that can maintain therapeutic microparticles in suspension for 30 minutes or more. Summary of the Invention

[0007] The present disclosure provides a formulation for intravenous injection, comprising, based on the total weight of the formulation, about 0.80 weight percent to about 1.60 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation, about 0.12 weight percent to about 0.28 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) polymer, based on the weight of the formulation, about 0.22 weight percent to about 0.78 weight percent mannitol, based on the total weight of the formulation, and water.

[0008] In some embodiments, the formulation comprises about 1.19 weight percent to about 1.54 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation, or about 1.33 weight percent to about 1.47 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation. In some embodiments, the formulation comprises about 0.19 weight percent to about 0.21 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) polymer, based on the total weight of the formulation. In some embodiments, the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) has a weight average molecular weight of about 8,400 daltons. In certain embodiments, the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) has the structure of Formula I: [ka] In the formula, X is 72 to 88, Y is 23 to 30, and Z is 72 to 88.

[0009] In some embodiments, the formulation comprises about 0.48 weight percent to about 0.66 weight percent mannitol, or about 0.49 weight percent to about 0.63 weight percent mannitol, based on the total weight of the formulation. In some embodiments, the formulation further comprises therapeutic microparticles. In some embodiments, the therapeutic microparticles comprise an active agent, a polymer-active agent conjugate, or a combination thereof. In some embodiments, the polymer-active agent conjugate comprises a polymeric carrier conjugated to an active agent. In some embodiments, the formulation comprises about 500 million therapeutic microparticles per milliliter of formulation to about 3 billion therapeutic microparticles per milliliter of formulation. In some embodiments, the formulation comprises about 1 billion therapeutic microparticles per milliliter of formulation to about 2.5 billion therapeutic microparticles per milliliter of formulation. In certain embodiments, the therapeutic microparticles have a longest dimension of about 1,700 nm to about 3,000 nm, a shortest dimension of about 100 nm to about 200 nm, or a combination thereof. In some embodiments, the therapeutic microparticles are disc-shaped with a diameter of about 900 nm to about 2,800 nm and a thickness of about 200 nm to about 800 nm. In some embodiments, the polymeric carrier comprises poly-L-glutamic acid, poly(lactic acid), poly(glycolic acid), poly(D-lactic acid-co-glycolic acid), poly(L-lactic acid-co-glycolic acid), poly(D,L-lactic acid-co-glycolic acid), poly(caprolactone), poly(valerolactone), poly(hydroxybutyric acid), poly(hydrovaleric acid), polydioxinanone, and combinations thereof. In some embodiments, the active agent comprises a drug, a bioactive compound, a chemotherapeutic agent, an immunosuppressant, a cytokine, a cytotoxic agent, a nucleolytic compound, an imaging agent, a prodrug enzyme, a gene, a nucleic acid, shRNA, siRNA, a DNA fragment, an RNA fragment, a plasmid, or any combination thereof. In some embodiments, the therapeutic microparticles are porous silicon microparticles comprising a polymer of L-glutamic acid conjugated to doxorubicin, the polymer having a weight average molecular weight of about 50 kDa to about 100 kDa, including about 70 kDa to about 80 kDa.

[0010] The present disclosure further provides methods for preparing a formulation for intravenous injection. In some embodiments, the method comprises adding sodium carboxymethylcellulose, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), and mannitol to water, either simultaneously or in any order, to form a formulation. In some embodiments, the formulation comprises about 0.80 weight percent to about 1.60 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation; about 0.12 weight percent to about 0.28 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), based on the total weight of the formulation; and about 0.22 weight percent to about 0.78 weight percent mannitol, based on the total weight of the formulation. In some embodiments, the method further comprises adding therapeutic microparticles to the formulation to form a pharmaceutical formulation, wherein the therapeutic microparticles comprise polymeric doxorubicin-containing porous silicon microparticles or siRNA-containing porous silicon microparticles. In some embodiments, the method further comprises adding therapeutic microparticles to the formulation to form a pharmaceutical formulation, wherein the therapeutic microparticles comprise porous silicon microparticles comprising an active agent, a polymer-active agent conjugate, or a combination thereof. In some embodiments, the polymer-active agent conjugate comprises a polymer carrier conjugated to an active agent. In some embodiments, the method further comprises adding therapeutic microparticles to the formulation to form a pharmaceutical formulation, wherein the therapeutic microparticles are porous silicon microparticles comprising a polymer of L-glutamic acid conjugated to doxorubicin, the polymer having a weight average molecular weight of about 50 kDa to about 100 kDa, including about 70 kDa to about 80 kDa. In some embodiments, the polymer carrier comprises poly-L-glutamic acid, poly(lactic acid), poly(glycolic acid), poly(D-lactic acid-co-glycolic acid), poly(L-lactic acid-co-glycolic acid), poly(D,L-lactic acid-co-glycolic acid), poly(caprolactone), poly(valerolactone), poly(hydroxybutyric acid), poly(hydrovaleric acid), polydioxynanone, or a combination thereof.In some embodiments, the active agent comprises a drug, a bioactive compound, a chemotherapeutic agent, an immunosuppressant, a cytokine, a cytotoxic agent, a nucleolytic compound, an imaging agent, a prodrug enzyme, a gene, a nucleic acid, shRNA, siRNA, a DNA fragment, an RNA fragment, a plasmid, or any combination thereof. In some embodiments, the method further comprises adding therapeutic microparticles to the formulation before, during, or after adding sodium carboxymethylcellulose, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), or mannitol to the water to form a pharmaceutical formulation. In some embodiments, the method further comprises forming a suspension of therapeutic molecules prior to adding therapeutic microparticles, i.e., porous silicon microparticles containing polymers of L-glutamic acid conjugated with doxorubicin, to the formulation to form the pharmaceutical formulation, wherein forming the suspension of therapeutic microparticles further comprises dissolving a doxorubicin solution containing poly(L-glutamic acid) polymer conjugated with doxorubicin in a liquid to form a doxorubicin solution, the liquid comprising about 50 weight percent to 100 weight percent methanol based on the total weight of the doxorubicin solution, and contacting the doxorubicin solution with the porous silicon microparticles to form the therapeutic microparticles. In some embodiments, after forming the suspension of pharmaceutical particles and prior to forming the pharmaceutical formulation, the method further comprises subjecting the doxorubicin solution to centrifugal force to separate a supernatant from an aliquot of the doxorubicin solution, and removing the supernatant. In some embodiments, the method further comprises applying vibration, ultrasound, or a combination thereof to agitate the doxorubicin solution after forming the suspension of pharmaceutical particles and before forming the pharmaceutical formulation.

[0011] The present disclosure further provides methods for administering therapeutic microparticles to a subject in need thereof. In some embodiments, the methods for administering therapeutic microparticles to a subject include providing a pharmaceutical formulation comprising, based on the total weight of the formulation, about 0.80 weight percent to about 1.60 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation, about 0.12 weight percent to about 0.28 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) polymer, based on the total weight of the formulation, about 0.22 weight percent to about 0.78 weight percent mannitol, based on the total weight of the formulation, and water; placing the pharmaceutical formulation into an intravenous line; and connecting the intravenous line to the subject. In some embodiments, the therapeutic microparticles comprise an active agent, a polymer-active agent conjugate, or a combination thereof, wherein the polymer-active agent conjugate comprises a polymer carrier conjugated to an active agent. In some embodiments, the pharmaceutical formulation comprises about 500 million therapeutic microparticles per milliliter to about 3 billion therapeutic microparticles per milliliter of the pharmaceutical formulation.

[0012] The above summary, as well as the following detailed description of the embodiments, may be better understood when read in conjunction with the accompanying drawings. For purposes of illustration, the drawings show some embodiments that may be preferred. It is understood that the embodiments shown are not limited to the precise details shown. Unless otherwise noted, the drawings are not to scale. [Brief explanation of the drawings]

[0013] [Figure 1] 2A-2D are graphs showing the effect of rotational flow on suspension time of therapeutic microparticles (pDox) in certain embodiments of the formulation. [Figure 2] FIG. 1 shows the suspension time of therapeutic microparticles (MSV / siRNA) in one embodiment of a formulation of the present disclosure compared to water. DETAILED DESCRIPTION OF THE INVENTION

[0014] Unless otherwise stated, all measurements are in standard metric units.

[0015] Unless otherwise specified, all instances of the words "a," "an," or "the" may refer to one or more of the words they modify.

[0016] Unless otherwise specified, the term "about" refers to ±10% of a stated non-percentage number, rounded to the nearest number to the stated precision. For example, about 105.3 mm includes 94.8 to 115.8 mm. Unless otherwise specified, the term "about" refers to a percentage number ±5%. For example, about 20% can include 15 to 25%. When the term "about" is used in connection with a range, it refers to an appropriate amount less than the lower limit and greater than the upper limit. For example, about 100 mm to about 200 mm would include 90 mm to 220 mm.

[0017] Unless otherwise specified, when a range of values ​​refers to values ​​that are readily and routinely changed in a laboratory environment, such as weight, mass, concentration, temperature, or pressure, the range of values ​​or measurements includes all values ​​within that range. For example, the range 0.80% by weight to 1.60% by weight includes 0.9, 0.95, 1.0, 1.1, 1.2, 1.25, 1.5, 1.59, and all subranges therebetween.

[0018] Unless otherwise specified, the terms "for example" or "eg," as used herein, are for illustrative purposes only and should not be construed as limiting the disclosure to only those matters expressly mentioned herein.

[0019] Unless otherwise specified, the term "microparticle" means a particle having a size between 0.1 microns and 100 microns.

[0020] Unless otherwise specified, the term "micron" means "micrometer" and the term "microns" means "micrometers." These terms are interchangeable.

[0021] As used herein, the term "subject" (also interchangeably referred to as "host" or "patient") refers to any subject capable of receiving one or more of the pharmaceutical formulations disclosed herein. In some embodiments, the subject is a vertebrate, and is intended to refer to any animal species, including mammalian species such as humans. In certain embodiments, "subject" refers to any animal host, including, but not limited to, any mammalian host, including humans. In some embodiments, the term refers to any mammalian host, including humans and non-human primates, bovines, canines, caprines, cabines, corvids, epines, equines, felines, hircines, lapines, leporines, wolves, murines, ovines, swine, frogs, racines, and foxes, including, but not limited to, livestock, animal laboratory specimens, exotic animals, and companion animals, pets, and any animals under veterinary care. Mammalian species that can benefit from the methods of the present disclosure include, but are not limited to, humans, non-human primates, such as apes, chimpanzees, monkeys, and orangutans, domestic animals including dogs and cats, as well as livestock such as horses, cows, pigs, sheep, and goats, or other mammalian species such as, but not limited to, mice, rats, guinea pigs, rabbits, and hamsters. The subject can be a patient of any age capable of mounting an immune response and responding to a drug or vaccination. In certain embodiments, the mammalian patient is a human.

[0022] Unless otherwise specified, the term "naturally occurring" as used herein when applied to an object refers to the fact that the object is one that can be found in nature.

[0023] Unless otherwise specified, the term "non-naturally occurring," as used herein, when applied to an object, refers to an object that has been created by human action and that has a structure, composition, or combination thereof that is not found in nature without human intervention, or that is not found in nature without human intervention. For example, a polypeptide found in nature that has a structure that has been modified in a laboratory is a non-naturally occurring polypeptide.

[0024] Unless otherwise specified, the term "polypeptide," as used herein, is intended to include a singular "polypeptide" and plural "polypeptides," and includes any chain(s) of two or more amino acids. Accordingly, as used herein, terms including, but not limited to, "peptide," "dipeptide," "tripeptide," "protein," "enzyme," "amino acid chain," and "contiguous amino acid sequence" are all included within the definition of the term "polypeptide," and the term "polypeptide" may be used in place of or interchangeably with any of these terms. The term further includes polypeptides that have undergone one or more post-translational modification(s), such as, for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translational processing, or modification by incorporation of one or more unnatural amino acids.

[0025] Unless otherwise specified, conventional nomenclature in the art for polynucleotide and polypeptide structures is used herein. For example, to describe amino acids, one-letter and three-letter abbreviations are commonly used: alanine (A, Ala), arginine (R, Arg), asparagine (N, Asn), aspartic acid (D, Asp), cysteine ​​(C, Cys), glutamine (Q, Gln), glutamic acid (E, Glu), glycine (G, Gly), histidine (H, His), isoleucine (I, Ile), leucine (L, Leu), methionine (M, Met), phenylalanine (F, Phe), proline (P, Pro), serine (S, Ser), threonine (T, Thr), tryptophan (W, Trp), tyrosine (Y, Tyr), valine (V, Val), and lysine (K, Lys). The amino acid residues described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue as long as the desired properties of the polypeptide are retained.

[0026] Unless otherwise specified, the term "sequence" when referring to amino acids relates to a given amino acid chain, e.g., all or part of the linear N-terminal to C-terminal order of given amino acids in a polypeptide or protein, and "subsequence" means any contiguous stretch of amino acids in a sequence, e.g., at least three contiguous amino acids in a given protein or polypeptide sequence. With respect to nucleotide chains, "sequence" and "subsequence" have similar meanings in reference to the 5' to 3' order of nucleotides.

[0027] Unless otherwise specified, the terms "provide," "provided," or "providing" refer to any supplying, producing, purchasing, manufacturing, constructing, forming, selecting, shaping, converting, introducing, adding, or incorporating any method or system of any embodiment herein.

[0028] Unless otherwise specified, all reactions, procedures, and preparations are or can be carried out at normal temperature and pressure, i.e., 20° C. and 1 atmosphere.

[0029] Unless otherwise specified, properties (height, width, length, ratios, etc.) described herein are understood to be averaged measurements.

[0030] Advances are being made in the field of nanomedicine. Researchers are increasingly inventing methods for designing and fabricating micro- and nanoscale platforms for delivering active agents, such as drugs and biomaterials. However, for nanomedicine to be adopted and utilized in modern healthcare settings, methods must be found to adapt nanomedicine classes, such as therapeutic microparticles, to existing and approved modern medicines. A constant challenge in the application of therapeutic microparticles is the need to ensure that these therapeutic microparticles do not aggregate and settle in solution before they can be used. This challenge becomes even more difficult when adapting therapeutic microparticles for use in modern and common treatments, such as intravenous therapy or intravenous administration of therapeutic agents. If therapeutic agents aggregate or settle before or during IV administration, patients may be injured or killed.

[0031] In practice, most IV drugs are dispensed by hospital or laboratory pharmacists. Therefore, realistically, IV drugs must wait 30 minutes to an hour before they can be picked up and administered to a patient by a healthcare professional. Once administered to a patient, IV drugs are typically administered via pump or gravity at a rate set by the healthcare professional. Depending on the wait time and rate, IV drugs must wait 60 to 90 minutes before the entire drug can be injected into the patient's vein. Naturally, no healthcare professional or hospital administrator wants to risk the therapeutic agent clumping or settling at the 90-minute limit, especially when this could result in patient injury or death. The longer an IV drug can keep the therapeutic particles safely suspended in solution, the better, safer, and more readily available the IV drug product and method of use will be.

[0032] If all of the above weren't daunting enough, pharmacists also face the challenge of not being able to simply use any ingredients to create an IV formulation. Instead, they can only use compounds and liquids that are approved by the Food and Drug Administration (FDA) for use in IV formulations.

[0033] Furthermore, because the goal is an IV formulation, simply any microparticle can be used as a platform for holding the therapeutic agent. For example, if the microparticle is too large or has too rough a shape, the therapeutic microparticle increases the likelihood of injury upon introduction into the patient. Conversely, if the microparticle used to hold the therapeutic agent is too small, it may cause unpredictable side effects, such as bypassing the blood-brain barrier.

[0034] The disclosed IV formulation has been found to overcome all of these challenges by providing an IV formulation capable of stably suspending therapeutic microparticles for periods of time, such as 105 minutes and 2.5 hours, using only FDA-approved compounds and liquids in the IV formulation. Furthermore, it has been found that there are specific concentrations of compounds and liquids in the IV formulations disclosed herein that can provide this stability, and that concentrations outside these ranges will lose their suspension state much more quickly. Next, it has been found that the formulation is compatible with therapeutic microparticles, and that basic properties of the microparticles, such as size and shape distribution, can be controlled by microfabrication. Because the formulation is compatible with therapeutic microparticles and the therapeutic agents themselves are diverse, it has also been found that the IV formulation should be compatible and stabilize a wide range of therapeutic microparticles, regardless of the specific therapeutic agent contained within the microparticles.

[0035] The present disclosure provides formulations for intravenous injection. In some embodiments, the formulation for intravenous injection comprises about 0.80 weight percent to about 1.60 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation. In some embodiments, the formulation comprises about 0.12 weight percent to about 0.28 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), based on the total weight of the formulation. In some embodiments, the formulation comprises about 0.22 weight percent to about 0.78 weight percent mannitol, based on the total weight of the formulation, and water.

[0036] In some embodiments, the formulation comprises about 1.19 weight percent to about 1.54 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation. In some embodiments, the formulation comprises about 1.33 weight percent to about 1.47 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation. In some embodiments, the formulation comprises about 0.90 weight percent to about 1.5 weight percent carboxymethylcellulose, including about 1.0 weight percent to about 1.45 weight percent or about 1.0 weight percent to about 1.35 weight percent sodium carboxymethylcellulose. It has been found that when the formulation contains more than about 1.60 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation, the formulation is unable to suspend therapeutic microparticles after 90 minutes of incubation. It has been found that when the formulation contains less than about 0.80 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation, the formulation is unable to suspend therapeutic microparticles after 90 minutes of incubation.

[0037] In some embodiments, the formulation comprises about 0.19 weight percent to about 0.21 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), based on the total weight of the formulation. In some embodiments, the formulation comprises poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) in an amount of about 0.13 weight percent to about 0.26 weight percent. In some embodiments, the formulation comprises poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) in an amount of about 0.14 weight percent to about 0.25 weight percent, including about 0.17 weight percent to about 0.23 weight percent, and including about 0.18 weight percent to about 0.22 weight percent. It has been found that above about 0.21 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), based on the total weight of the formulation, the formulation is unable to suspend therapeutic microparticles for more than 90 minutes. Below about 0.19 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(polyethylene glycol), based on the total weight of the formulation, the formulation is unable to suspend therapeutic microparticles for more than 90 minutes.

[0038] In some embodiments, the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) has a weight average molecular weight of about 8,400 Daltons.

[0039] In some embodiments, the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) has the structure of Formula I: [ka] wherein X is 72-88, Y is 23-30, and Z is 72-88. In some embodiments, X is 75-85, including 78-82, Y is 25-28, including 26-28, and Z is 75-85, including 78-82. In some embodiments, the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) is Poloxamer 188 (also known as P188) (CAS 9003-11-6), which is commercially available from SIGMA-ALDRICH®, among others.

[0040] In some embodiments, the formulation comprises about 0.48 weight percent to about 0.66 weight percent mannitol, based on the total weight of the formulation. In some embodiments, the formulation comprises about 0.49 weight percent to about 0.63 weight percent mannitol, based on the total weight of the formulation. In some embodiments, the formulation comprises about 0.28 weight percent to about 0.75 weight percent mannitol, including about 0.30 weight percent to about 0.72 weight percent, or about 0.35 weight percent to about 0.70 weight percent, or about 0.40 weight percent to about 0.68 weight percent, or about 0.45 weight percent to about 0.65 weight percent. It has been found that if the amount of mannitol in the formulation exceeds about 0.66 weight percent, the formulation is unable to suspend therapeutic microparticles for more than 90 minutes. It has been found that if the formulation contains less than about 0.48 weight percent mannitol, based on the total weight of the formulation, the formulation is unable to suspend therapeutic microparticles for more than 90 minutes.

[0041] Although the formulations disclosed herein are intended for suspending therapeutic microparticles in an IV formulation, the components of the formulation are sufficiently stable that they can be shipped and stored for several months before adding any therapeutic microparticles. Thus, in some embodiments, formulations for intravenous injection can be manufactured and sold without or excluding microparticles, such as therapeutic microparticles, allowing for the rapid and easy formulation of IV formulations containing therapeutic microparticles by simply mixing the therapeutic microparticles immediately before use.

[0042] In some embodiments, the formulation further comprises a therapeutic microparticle, the therapeutic microparticle comprising an active agent, a polymer-active agent conjugate, or a combination thereof. The polymer-active agent conjugate comprises a polymer carrier conjugated to the active agent.

[0043] In some embodiments, the polymer carrier comprises poly-L-glutamic acid, poly(lactic acid), poly(glycolic acid), poly(D-lactic acid-co-glycolic acid), poly(L-lactic acid-co-glycolic acid), poly(D,L-lactic acid-co-glycolic acid), poly(caprolactone), poly(valerolactone), poly(hydroxybutyric acid), poly(hydrovaleric acid), polydioxynanone, and combinations thereof.

[0044] In some embodiments, the active agent includes a drug, a bioactive compound, a chemotherapeutic agent, an immunosuppressant, a cytokine, a cytotoxic agent, a nucleolytic compound, an imaging agent, a prodrug enzyme, a gene, a nucleic acid, an shRNA, an siRNA, a DNA fragment, an RNA fragment, a plasmid, or any combination thereof. The active agent can be any physiologically or pharmacologically active substance that produces a desired biological effect. The active agent can be naturally occurring or can be produced by synthetic or recombinant methods, or a combination thereof. The active agent can be a cancer chemotherapeutic agent. For a more detailed description of anticancer drugs and other active agents, those skilled in the art can refer to several instruction manuals, such as, but not limited to, the Physician's Desk Reference and Goodman & Gilman's *The Pharmacological Basis of Therapeutics*, Tenth edition, Hardman, JG et al. (eds.), McGraw-Hill Professional (2001).

[0045] Exemplary agents include, but are not limited to, one or more anti-cancer drugs, or one or more combinations thereof, of conventional drugs such as camptothecin, docetaxel, temozolomide, carmustine, paclitaxel, gemcitabine, and anthracyclines (including, but not limited to, doxorubicin, liposomal doxorubicin, daunorubicin, etc.).

[0046] Exemplary chemotherapeutic agent(s) and immunosuppressant(s) include 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), altretamine, arsenic trioxide, asparaginase, azacitidine, azathioprine, bendamustine, bleomycin, busulfan, cabazitaxel, capecitabine (Xeloda), carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clorambucil, cisplatin, clorambucil ... Farabine, cyclophosphamide, cytarabine (Ara-C), cyclosporine, dacarbazine, dactinomycin, daunorubicin, decitabine, dexamethasone, docetaxel, doxorubicin (adriamycin), doxorubicin liposome, epirubicin, eribulin, etoposide (VP-16), floxuridine, fludarabine, gemcitabine (Gemzar), hydroxyurea, idarubicin, Ifosfamide, irinotecan, irinotecan liposomal, ixabepilone, lomustine, mechlorethamine, melphalan, methotrexate, methylprednisolone, mitomycin-C, mitotane, mitoxantrone, mycophenolate, nab-paclitaxel, nelarabine, omacetaxine, oxaliplatin, paclitaxel, pegaspargase, pemetrexed (Alimta), pentostatin, protease inhibitors These include, but are not limited to, laratrexate, prednisone, procarbazine, rapamycin, romidepsin, streptozocin, tacrolimus, temozolomide, teniposide, thioguanine, thiotepa, tipiracil, topotecan, trabectedin, trifluridine, valrubicin, vinblastine, vincristine, vincristine liposomal, vinorelbine, vorinostat, and any combination thereof.

[0047] Exemplary cytokine(s) include, but are not limited to, lymphokines, monokines, traditional polypeptide hormones, and mixtures thereof.

[0048] Exemplary cytotoxic agent(s) include, but are not limited to, altretamine, trabectedin, busulfan, carmustine, lomustine, doxorubicin, valrubicin, bleomycin, dactinomycin, methotrexate, floxuridine, clofarabine, pralatrexate, vinblastine, vinorelbine, vincristine, vindesine, cisplatin, teniposide, etoposide, topotecan, irinotecan, and any combination thereof.

[0049] Exemplary nucleolytic agent(s) include, but are not limited to, Dnase I, Exonuclease III, mung bean nuclease, S1 nuclease, RNAse H, or Rnase A, or chemical compounds such as hydrogen peroxide, osmium tetroxide, hydroxylamine, or potassium permanganate, or chemical conditions such as high or low pH, and any combination thereof.

[0050] Exemplary contrast agent(s) include, but are not limited to, Gd(III)-based agents, Mn(II)-based agents, and mixtures thereof. Gd(III) contrast agents include any Gd(III)-based contrast agent, such as gadobenic acid, gadobutrol, gadocholenic acid, gadodenterate, gadodiamide, gadofosveset, gadomelitol, gadopenamide, gadopentetic acid, gadoteric acid, gadoversemide, gadoxetic acid, or pharmaceutically acceptable salts thereof, and mixtures thereof. In another embodiment, the contrast agent may include Gd(III) ions within carbon-based particles. For example, Gd(III)-CA may include gadofullerene or gado nanotubes. Furthermore, the gado nanotubes may be bundled or unbundled. In some embodiments, the contrast agent is a Mn(II)-based contrast agent (Mn-CA), which is known in the art. In some embodiments, the imaging agent may include Magnevist (MAG) and / or Dotarem. In further embodiments, the imaging agent may be a pharmaceutically acceptable salt of the imaging agents listed above.

[0051] In some embodiments, the porous particles of the present disclosure should have a relatively large porosity to allow loading of the active agent inside the pores of the porous particles. In some embodiments, the porous particles of the present disclosure can optionally be coated with a targeting moiety. Such embodiments are useful for targeted delivery of the active compound to a desired disease site.

[0052] Exemplary bioactive compound(s) include, but are not limited to, peptides, proteins, therapeutic agents, diagnostic agents, non-biological materials, and combinations thereof. The active agent can be any physiologically or pharmacologically active substance capable of exerting a desired biological effect. In some embodiments, the therapeutic agent is an siRNA or microRNA that silences one or more genes expressing cancer cells or tumors. The therapeutic agent can also be utilized to genetically manipulate the genomes of cancer cells and / or stromal cells within a tumor, such as with the CRISPR / Cas9 system. In some embodiments, the therapeutic agent includes, but is not limited to, one or more proteins, peptides, polypeptides (including but not limited to, enzymes, antibodies, antigens, antigen-binding fragments, etc.), RNA molecules (including but not limited to, siRNA, microRNA, iRNA, mRNA, tRNA, or catalytic RNA, e.g., ribozymes), DNA molecules (including but not limited to, oligonucleotides, polynucleotides, genes, coding sequences (CDS), introns, exons, plasmids, cosmids, phagemids, baculoviruses, vectors (including but not limited to, viral vectors, virions, viral particles, etc.), peptide nucleic acids, detection agents, imaging agents, contrast agents, detectable gases, radionuclides, etc., or one or more additional chemotherapeutic agents, surgical intervention (e.g., tumor resection), radiation therapy, etc., or any combination thereof, as part of a multifactorial or multifocal treatment plan for the affected patient. In some embodiments, the therapeutic agent includes, but is not limited to, an anti-cancer agent, an anti-tumor agent, agents), antineoplastic or cytotoxic agents, transcription factors, immunomodulators, immunostimulators, neuroactive agents, anti-inflammatory agents, chemotherapeutic agents, hormones, so-called "trophic factors," chemokines, receptor agonists or antagonists, etc., or any combination thereof.

[0053] Exemplary nucleic acid(s) include, but are not limited to, polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base (including abasic sites). In some embodiments, nucleic acids comprise polymers of ribonucleosides or deoxyribonucleosides, typically covalently linked by phosphodiester bonds between the subunits, but in some cases by phosphorothioates, methylphosphonates, etc. In some embodiments, nucleic acids include single- and double-stranded DNA, and single- and double-stranded RNA. Exemplary nucleic acids include, but are not limited to, gDNA, hnRNA, mRNA, rRNA, tRNA, microRNA (miRNA), small interfering RNA (siRNA), intranucleolar RNA (snORNA), small nuclear RNA (snRNA), and small transient RNA (stRNA), and the like, and mixtures thereof.

[0054] In some embodiments, the active agent can be selected from the group consisting of a gene, a nucleic acid, an shRNA, an siRNA, a microRNA, a DNA fragment, an RNA fragment, a plasmid, and combinations thereof. In exemplary embodiments, the active agent is an siRNA or a microRNA that silences one or more genes expressed by cancer cells or tumors. The active agent can also be used to genetically manipulate the genome of cancer cells and / or stromal cells within a tumor, such as with the CRISPR / Cas9 system.

[0055] In some embodiments, the therapeutic microparticles are porous silicon microparticles that store an active agent, polymer-active agent conjugates, or combinations thereof. In some embodiments, the polymer-active agent conjugates can include a polymer carrier conjugated to an active agent.

[0056] Disclosed herein are methods for creating porous microparticles. In some embodiments, the method for creating porous microparticles can include providing a substrate, forming a porous layer on the surface of the substrate, patterning one or more microparticles on the substrate, and releasing the microparticles from the substrate, with each released microparticle comprising a portion of the porous layer. In some embodiments, the formation of the porous layer can occur before the patterning step, and in some embodiments, the formation of the porous layer can occur subsequent to the patterning step. In some embodiments, the method can employ micro / nanofabrication techniques to control the size of the particles formed. Some advantages of using nanofabrication techniques include: 1) the ability to create particles with a variety of predetermined shapes, such as, but not limited to, discoids, spheres, pyramidal, cubic, or rectangular shapes; 2) highly precise dimensional control; 3) control of porosity and pore profile; and 4) the ability to perform complex surface modifications while still using conventional and readily available methods. In some embodiments, the microparticles have pores for filling or internalization.

[0057] In some embodiments, the substrate may be composed of any of a number of materials. In some embodiments, the substrate may have at least one planar surface on which one or more particles may be patterned. In some embodiments, the substrate comprises a wet-etchable material, i.e., a material that can be made porous by wet etching techniques such as electrochemical etching.

[0058] In certain embodiments, the substrate may be a crystalline substrate, such as a wafer, a semiconductor wafer, or a silicon wafer. In certain embodiments, the substrate may be a semiconductor substrate, i.e., a substrate comprising one or more semiconductor materials. Examples of semiconductor materials include, but are not limited to, Ge, GaAs, InP, SiC, GaP, and GaN. In many embodiments, silicon may be the substrate material. Substrate properties, such as doping level, resistivity, and surface crystalline orientation, can be selected to obtain the desired size, density, and patterning of the pores.

[0059] In some embodiments, the porous layer can be formed on the substrate using a number of techniques. In some embodiments, the porous layer is formed using a wet etching technique, i.e., by exposing the substrate to an etchant containing at least one etchant, such as a strong acid. In some embodiments, the specific etchant depends on the material of the substrate. For example, for a germanium substrate, the etchant may be hydrochloric acid (HCl), while for a silicon substrate, the etchant may be hydrofluoric acid (HF). In some embodiments, the formation of the porous layer is carried out using an electrochemical etching process, during which an etching current is passed through the substrate. Electrochemical etching of silicon substrates to form porous silicon layers is described in detail, for example, in Salonen et al., "Mesoporous Silicon in Drug Delivery Applications," Journal of Pharmaceutical Sciences, 97(2):632-653 (2008). For electrochemical etching of silicon substrates, the etchant may include, in addition to HF, water, ethanol, or a combination or mixture thereof.

[0060] In some embodiments, during the electrochemical etching process, the substrate can function as one of the electrodes. For example, during electrochemical etching of silicon, the silicon substrate can function as the anode, and the cathode can be an inert metal such as platinum (Pt). In such cases, a porous layer is formed on the side of the substrate facing away from the inert metal cathode. In some other embodiments, during electrochemical etching, the substrate is placed between two electrodes, each of which can include an inert metal.

[0061] In some embodiments, the electrochemical etching process can be carried out in a reactor or cell that is resistant to the etchant. For example, if the etchant is HF, the electrochemical etching process can be carried out in a reactor or cell that includes an HF-resistant material. An example of an HF-resistant material is a fluoropolymer, such as polytetrafluoroethylene. In some embodiments, the electrochemical etching can be carried out by monitoring the current at one electrode, e.g., the anodic current (galvanostatically) or the voltage (potentially). In some embodiments, the method can be carried out by electrochemical etching at a constant current density, which allows for better control over the properties of the formed porous layer and / or allows for better reproducibility between samples.

[0062] In some embodiments, if it is desired to form two different stable porous regions, two different constant currents can be applied, e.g., a first current density can be applied to form a first stable porous layer, and then a second current density can be applied to form a second stable porous layer, which differs from the first stable porous layer in pore size and / or porosity.

[0063] In some embodiments, parameters of the formed porous layer, such as pore size, porosity, thickness, pore profile and / or pore shape, and then the respective parameters of the produced particles, may be adjusted by selecting, for example, parameters of the electrochemical etching process, such as the concentration and composition of the etchant, the applied current (and potential), the etching time, the temperature, the stirring conditions, the presence or absence of illumination (and illumination parameters such as intensity and wavelength), and parameters of the etched substrate, such as the composition of the substrate, the resistivity of the substrate, the crystallographic orientation of the substrate, and the level and type of doping of the substrate.

[0064] In some embodiments, the pores of the formed porous layer may have a predetermined longitudinal profile along the length, which is a profile perpendicular or substantially perpendicular to the surface of the substrate. Such a longitudinal profile may be generated by varying the current density during electrochemical etching. For longitudinal pores of the porous layer, both the porosity and the pore size may vary. Thus, in some embodiments, the profiled pores in the porous layer and the produced porous particle may have small pores at the top, i.e., at the surface of the substrate, and large pores at the bottom, i.e., deeper in the substrate. In certain embodiments, the profiled pores in the porous layer and the produced porous particle may have large pores at the top and small pores at the bottom. In some embodiments, the profiled pores in the porous layer and the produced particle may also have different porosities at the top and bottom.

[0065] In some embodiments, the electrochemical etching can begin with a short, high current pulse to prevent or reduce the formation of a nucleation layer. Alternatively, the nucleation layer can be etched away after the porous layer is formed. Such etching can be performed by dry etching techniques, such as reactive ion etching (RIE). In certain embodiments, appropriate measures can be taken to protect the underlying region. For example, photoresist can be placed on the surface and planarized by baking, followed by plasma etching back to expose a portion of the substrate surface to be etched.

[0066] For electrochemical etching, the backside of the substrate, i.e., the side of the substrate opposite the side on which the porous layer is formed, can be coated with a conductive layer, such as a metal layer, to ensure electrical contact. Such conductive layers can be coated using a number of techniques, including thermal evaporation and sputtering.

[0067] In some embodiments, during electrochemical etching, the etchant can initiate its pore formation through the formation of a nucleation layer, which is a surface layer of the substrate in which the pores have properties that differ from the desired properties of the porous layer. In some embodiments, the nucleation layer can be characterized by irregularities in its pore properties and associated surface roughness, which can be on a scale larger than the pore size.

[0068] In some applications, the nucleation layer on the surface of the porous particles is undesirable. For example, when silicon porous particles are used to support smaller particles inside them, the nucleation layer on the surface of the larger ones can reduce the loading efficiency.

[0069] In some embodiments, the nucleation layer is removed or prevented from forming. In some embodiments, during electrochemical etching, a larger current may be applied to prevent the formation of the nucleation layer before applying current to create the desired pores in the porous layer. Furthermore, in some embodiments, after the porous layer is formed, the nucleation layer may be removed by dry etching, such as reactive ion etching (RIE).

[0070] In some embodiments, patterning one or more particles on the surface of the substrate can be performed using any of a number of techniques. In some embodiments, patterning can be performed using lithography techniques such as photolithography, X-ray lithography, deep UV lithography, nanoimprint lithography, or dip-pen lithography. Photolithography techniques can be, for example, contact aligner lithography, scanner lithography, or immersion lens lithography. In some embodiments, in the case of photolithography or molding, using different masks, it may be possible to design particles having several predetermined regular shapes, i.e., non-random shapes, such as spheres, squares, rectangles, ellipses, disks, and hemispheres. In some embodiments, patterning can be used to define the lateral shape and dimensions of the particles, i.e., the shape and dimensions of the particles in a cross section parallel to the surface of the substrate. In some embodiments, if the formation of a porous layer precedes patterning, the lateral dimensions of the produced particles are substantially the same as the lateral dimensions of the patterned portion. In some embodiments, if patterning is performed prior to the formation of the porous layer, the lateral dimensions of the produced particles may be larger than the lateral dimensions of the patterned portion. In some embodiments, patterning allows for the creation of particles having a predetermined, regular, i.e., non-random, lateral shape. For example, in photolithographic patterning, masks of various shapes can be used to create the desired predetermined shape, while in nanoimprint lithography, molds or stamps of various shapes can be used for the same purpose. In some embodiments, the predetermined, non-random lateral shape of the particles is not particularly limited. For example, the particles may be circular, square, polygonal, and elliptical. In some embodiments, the microparticles may be comprised of a shape selected from the group consisting of discoids, spheres, non-spheroids, oblate spheroids, and combinations thereof. In some embodiments, the microparticles are produced from porous or mesoporous silicon material that is discoidal in shape.

[0071] In some embodiments, the particles are released from the wafer after the patterning and porous layer formation steps by electropolishing, which involves applying a sufficiently high current density to the wafer. In some embodiments, releasing the particles from the wafer involves forming an additional porous layer having greater porosity than the already formed porous layer. In some embodiments, this more porous layer is referred to as the release layer. In some embodiments, the release layer can have a sufficiently large porosity so that it can be easily broken when desired, for example, using mechanical techniques such as exposing the substrate to ultrasonic energy. At the same time, the release layer can be strong enough to hold both the pre-formed porous layer and the substrate intact.

[0072] Any of a number of techniques can be used to modify the surface properties of particles, i.e., the surface properties of the particle's outer surface and / or the surface properties of the particle's pores. In some embodiments, surface modification of manufactured particles can be performed while the particle and substrate are still intact before the particles are released. In some embodiments, types of particle surface modification include, but are not limited to, chemical modification, including polymer modification and oxidation, plasma treatment, metal or metal ion coating, chemical vapor deposition (CVD) coating, atomic layer deposition (ALD), evaporated and sputtered films, and ion implantation. In some embodiments, the surface treatment is biological, for biomedical targeted and controlled degradation.

[0073] Since particle surface modification can be performed before the particles are released from the substrate, asymmetric surface modification is also possible in some embodiments. In some embodiments, asymmetric surface modification provides one side of the particle with a different surface modification than the other side of the particle. For example, one side of the particle's surface can be modified, while the other side of the particle's surface can remain unmodified. For example, the pores of the particle can be completely or partially filled with a sacrificial material, such as a sacrificial photoresist. Thus, during surface modification, only the outer surface of the particle is processed. After selectively removing the sacrificial material, only the outer surface of the particle is modified, i.e., the pore surfaces of the particle remain unmodified. In some embodiments, the outer surface can be patterned, for example, by photolithography, so that one portion of the outer surface can have one modification, while another portion of the outer surface can have a different modification. In some embodiments, the surface modification of the particle is performed by techniques including, but not limited to, oxidation, silanization, and binding of targeting moieties such as antibodies, and combinations thereof.

[0074] In some embodiments, the maximum characteristic size of the particles may be less than about 100 microns, or less than about 50 microns, or less than about 20 microns, or less than about 10 microns, or less than about 5 microns, or less than about 4 microns, or less than about 3 microns, or less than about 2 microns, or less than about 1 micron. In some embodiments, the maximum characteristic size of the particles may be between 500 nm and 3 microns, or between 700 nm and 2 microns. In some embodiments, the maximum characteristic size of the particles may be greater than about 2 microns, or greater than about 5 microns, or greater than about 10 microns.

[0075] In some embodiments, the microparticles have an average pore size of 1 micron or less, or 800 nm or less, or 500 nm or less, or 300 nm or less, or 200 nm or less, or 100 nm or less, or 80 nm or less, or 50 nm or less. In some embodiments, the microparticles may individually have an average pore size of 1 micron or less, or 800 nm or less, or 500 nm or less, or 300 nm or less, or 200 nm or less, or 100 nm or less, or 80 nm or less, or 50 nm or less. In some embodiments, the microparticles may have an average pore size of about 10 to about 60 nm or about 20 to about 40 nm.

[0076] In some embodiments, the therapeutic microparticles have a longest dimension of about 1,700 nm to about 3,000 nm, a shortest dimension of about 100 nm to about 200 nm, or a combination thereof, hi some embodiments, the therapeutic microparticles have a longest dimension of about 1,900 nm to about 2,800 nm, a shortest dimension of about 120 nm to about 170 nm, or a combination thereof.

[0077] In some embodiments, the therapeutic microparticles are disc-shaped with a diameter of about 900 nm to about 2,800 nm and a thickness of about 200 nm to about 800 nm. In some embodiments, the therapeutic microparticles are disc-shaped with a diameter of about 1000 to about 2,500 nm, including about 1200 nm to about 2,200 nm, or about 1400 nm to about 1,900 nm. In some embodiments, the therapeutic microparticles are disc-shaped with a thickness of about 300 nm to about 600 nm, or about 250 nm to about 700 nm, including about 350 nm to about 500 nm. An advantage of disc-shaped therapeutic microparticles is that their shape does not have sharp edges that could cause injury to a subject's circulatory system. An advantage of therapeutic particles with a diameter less than 2,800 nm is that the particles are less likely to clog a subject's arteries, veins, and capillaries.

[0078] In one embodiment, the therapeutic microparticles are porous silicon microparticles comprising a polymer of L-glutamic acid conjugated to doxorubicin, the polymer having a weight average molecular weight of about 50 kDa to about 100 kDa, including about 70 kDa to about 80 kDa.

[0079] Examples of therapeutic microparticles that may be used in the formulations of the present disclosure are disclosed in US10253424B2, which is incorporated herein by reference in its entirety.

[0080] siRNA oligonucleotides have been delivered to target cells using traditional delivery vehicles such as liposomes. Most siRNA products currently in clinical trials are packaged in either liposomes or lipid nanoparticles. Disease indications include liver-related metabolic diseases, liver cancer, or other cancers that have metastasized to the liver (Fitzgerald et al., "Effect of an RNA interference drug on the synthesis of proprotein convertase...Phase 1 trial," Lancet, 383:60-68 (2014)). Multistage vector (MSV) delivery systems are designed to maximize the delivery of therapeutic agents to tumor cells by sequentially crossing various biological barriers (Ferrari, M et al., "Vectoring siRNA therapeutics in the clinic," Nat. Rev. Clin. Oncol., 7(9):485-486 (2010)). This system consists of first-stage nanoporous silicon microparticles and second-stage liposome particles loaded within the nanopores of the first-stage microparticles. For gene silencing agent delivery, double-stranded RNA molecules are packaged within 30-40 nm liposomes, which are then loaded within 60-80 nm pores of nanoporous silicon microparticles (Shen, H. et al., "Delivery of gene silencing agents for breast cancer therapy," Breet Cancer Res., 15(3):205 (2013a); Tanaka, T et al., "Sustained small interfering RNA delivery by mesoporous silicon particles," Cancer Res., 70(9):3687-3696 (2009)). Once in the bloodstream, the first-stage microparticles travel with the bloodstream and settle in tumor vasculature, from which the liposomal siRNA is released. The release rate of siRNA is determined by the nanopore diameter, the liposome size, and the degradation rate of silicon.

[0081] In a typical MSV approach, charged nanoliposomes packaged with small molecule drugs or therapeutic siRNA are loaded inside the pores of pSi particles via electrostatic interactions and capillary forces. Once inside the body, the pSi particles (i.e., "Stage 1" particles) gradually degrade, and the nanoliposomes (i.e., "Stage 2" particles contained therein) are released from the Stage 1 pSi particles, thus facilitating multistage release. This delivery system has the advantages of both increasing loading efficiency and being easily tunable in terms of particle shape and size, allowing for efficient encapsulation of nanosized species in MSVs. This protects them from contact with unintended organs or cells, thus minimizing toxicity and improving efficacy. Multistage vector platforms for delivering siRNA to other tissues for cancer therapy have already been reported. See, for example, Shen, H et al., "Delivery of gene silencing agents for breast cancer therapy," Breast Cancer Res., 15(3):205(2013a) and Shen, H et al., "Enhancing chemotherapy response with sustained EphA2 silencing using multistage vector delivery," Clin. Cancer Res., 19(7):1806-1815(2013b).

[0082] Another example of a therapeutic microparticle that can be used in the formulation of the present disclosure is disclosed in US 2016 / 0369269 A1, namely, polycation-functionalized nanoporous silicon (PCPS) particles comprising (a) a population of first-stage particles of polycation-functionalized nanoporous silicon, and (B) a population of second-stage particles substantially contained within the population of first-stage particles, the second-stage particles comprising at least one active agent. These polycation-functionalized nanoporous silicon particles are capable of overcoming at least one biological barrier, such as one or more biological barriers selected from the group consisting of a blood rheological barrier, a reticuloendothelial barrier, a blood-brain barrier, a tumor-associated osmotic interstitial pressure barrier, an ionic or molecular pumping field barrier, a cell membrane barrier, an enzymatic degradation barrier, a nuclear membrane barrier, and combinations thereof. In US2016 / 0369269 A1, because the siRNA molecules are not pre-loaded into nanoparticles, it is demonstrated that they can therefore load much more siRNA (up to 100 times more nucleic acid) onto the same amount of porous silicon particles, thereby reducing the total amount of porous silicon required for each treatment. Less silicon means less toxicity. Furthermore, loaded PCPS particles can be dried and stored / transported for periods of days to weeks without significant loss of activity or efficacy.

[0083] In some embodiments, the therapeutic microparticles can have at least one targeting moiety on their surface that is specifically directed to target cells. In some embodiments, the at least one targeting moiety is selected from the group consisting of a ligand, an antibody, an antibody fragment, a peptide, an aptamer, a small molecule, and combinations thereof. For example, the ligand can be chemically linked to an appropriate reactive group on the particle surface. Protein ligands can be linked to amino-reactive groups and thiol-reactive groups under conditions effective to form thioether or amide bonds, respectively. Methods for attaching antibodies or other polymeric binders to inorganic or polymeric supports are described in detail elsewhere.

[0084] In some embodiments, the therapeutic microparticles optionally further comprise one or more additional components to aid, facilitate, or improve delivery of the prodrug and / or active metabolite contained therein, such as, but not limited to, one or more liposomes, lipid particles, lipid complexes, and optionally further comprise one or more binders, cytosurfactants, surfactants, lipid complexes, niosomes, ethosomes, transferosomes, phospholipids, sphingolipids, sphingosomes, or any combination thereof.

[0085] In certain embodiments, the formulation contains from about 500 million therapeutic microparticles per milliliter of the formulation to about 3 billion therapeutic microparticles per milliliter of the formulation. In some embodiments, the formulation contains from about 1 billion therapeutic microparticles per milliliter of the formulation to about 2.5 billion therapeutic microparticles per milliliter, including from about 1.2 billion therapeutic microparticles per milliliter to about 2.2 billion therapeutic microparticles per milliliter, or from about 1.4 billion therapeutic microparticles per milliliter to about 2.2 billion therapeutic microparticles per milliliter. In certain embodiments, the formulation contains from about 500 million therapeutic microparticles per milliliter of the formulation to about 1.5 billion therapeutic microparticles per milliliter of the formulation. An advantage of therapeutic microparticle concentrations of 300 million or less per milliliter of the formulation embodiments disclosed herein is that the formulation can keep the therapeutic microparticles suspended for at least one hour. Generally, the higher the concentration, the more difficult it is to maintain a suspension. An advantage of a therapeutic microparticle concentration of 500 million therapeutic microparticles per milliliter of formulation can be the ability to provide a high dose resulting in high efficacy in a subject. It has been found that when the therapeutic microparticle concentration exceeds 3 billion per milliliter of formulation, the microparticles tend to fall out of solution too quickly for use in IV therapy.

[0086] In some embodiments, the present disclosure further provides methods for preparing a formulation for intravenous injection. In some embodiments, the method includes adding sodium carboxymethylcellulose, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), and mannitol to water, either simultaneously or in any order, to form a formulation. In some embodiments, the method includes adding about 0.80 weight percent to about 1.60 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation. In some embodiments, the method includes adding about 0.12 weight percent to about 0.28 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), based on the total weight of the formulation. In some embodiments, the method includes adding about 0.22 weight percent to about 0.78 weight percent mannitol, based on the total weight of the formulation.

[0087] In some embodiments, the method further comprises adding therapeutic microparticles to the formulation to form a pharmaceutical formulation, hi some embodiments, the method comprises adding therapeutic microparticles comprising porous silicon microparticles containing polymeric doxorubicin or porous silicon microparticles containing siRNA.

[0088] In some embodiments, the method further comprises adding therapeutic microparticles to the formulation to form a pharmaceutical formulation. In some embodiments of the method, the therapeutic microparticles comprise porous silicon microparticles storing an active agent, a polymer-active agent conjugate, or a combination thereof. In some embodiments of the method, the polymer-active agent conjugate comprises a polymer carrier conjugated to an active agent.

[0089] In some embodiments, the method further comprises adding therapeutic microparticles to the formulation to form a pharmaceutical formulation. In some embodiments of the method, the therapeutic microparticles are porous silicon microparticles comprising a polymer of L-glutamic acid conjugated to doxorubicin. In some embodiments of the method, the polymer has a weight average molecular weight of about 50 kDa to about 100 kDa, including about 70 kDa to about 80 kDa.

[0090] In some embodiments, the polymeric carrier comprises poly-L-glutamic acid, poly(lactic acid), poly(glycolic acid), poly(D-lactic acid-co-glycolic acid), poly(L-lactic acid-co-glycolic acid), poly(D,L-lactic acid-co-glycolic acid), poly(caprolactone), poly(valerolactone), poly(hydroxybutyric acid), poly(hydrovaleric acid), polydioxynanone, or a combination thereof.

[0091] In some embodiments, the active agent comprises a drug, a bioactive compound, a chemotherapeutic agent, an immunosuppressant, a cytokine, a cytotoxic agent, a nucleolytic compound, an imaging agent, a prodrug enzyme, a gene, a nucleic acid, an shRNA, an siRNA, a DNA fragment, an RNA fragment, a plasmid, or any combination thereof.

[0092] In some embodiments, the method further comprises adding therapeutic microparticles to the formulation before, during, or after adding sodium carboxymethylcellulose, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), or mannitol to the water to form a pharmaceutical formulation.

[0093] In some embodiments, the method further comprises forming a suspension of therapeutic microparticles. In some embodiments, the method further comprises forming a suspension of therapeutic microparticles, wherein the therapeutic microparticles are porous silicon microparticles comprising polymers of L-glutamic acid conjugated with doxorubicin, prior to adding the therapeutic microparticles to a formulation to form the pharmaceutical formulation. In some embodiments, the suspension of therapeutic microparticles is formed by dissolving poly(L-glutamic acid) polymers conjugated with doxorubicin in a liquid to form a doxorubicin solution, the liquid comprising about 70 weight percent to 99 weight percent methanol, about 80 weight percent to 95 weight percent methanol, or about 50 weight percent to about 100 weight percent methanol, based on the total weight of the doxorubicin solution, and then contacting porous silicon microparticles with the doxorubicin solution to form the therapeutic microparticles. An advantage of forming therapeutic microparticles by contacting a doxorubicin solution with porous silicon microparticles in a liquid containing about 50 to 100 weight percent methanol may lie in the ability of this liquid to suspend the microparticles without inadvertently degrading the doxorubicin-conjugated poly(L-glutamic acid) polymer.

[0094] In some embodiments, after forming the suspension of pharmaceutical particles and before forming the pharmaceutical formulation, the method includes centrifuging the doxorubicin solution to remove the supernatant and separating the supernatant from an aliquot of the doxorubicin solution. In some embodiments, after forming the suspension of pharmaceutical particles and before forming the pharmaceutical formulation, the doxorubicin solution is agitated using vibration, ultrasound, or a combination thereof.

[0095] The present disclosure further provides methods of administering therapeutic microparticles to a subject in need thereof. In some embodiments, the methods of administering therapeutic microparticles to a subject include providing a pharmaceutical formulation comprising, based on the total weight of the formulation, about 0.80 weight percent to about 1.60 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation, about 0.12 weight percent to about 0.28 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), based on the total weight of the formulation, about 0.22 weight percent to about 0.78 weight percent mannitol, based on the total weight of the formulation, therapeutic microparticles, and water. In some embodiments, the methods of administering therapeutic microparticles to a subject further include adding the pharmaceutical formulation to an intravenous line and connecting the intravenous line to the subject.

[0096] In some embodiments, the therapeutic microparticles comprise an active agent, a polymer-active agent conjugate, or a combination thereof, where the polymer-active agent conjugate comprises a polymeric carrier conjugated to an active agent.

[0097] In certain embodiments, the formulation contains about 500 million therapeutic microparticles per milliliter of formulation to about 3 billion therapeutic microparticles per milliliter of formulation. In some embodiments, the formulation contains about 1 billion therapeutic microparticles per milliliter of formulation to about 2.5 billion therapeutic microparticles per milliliter of formulation, including about 1.2 billion therapeutic microparticles per milliliter to about 2.2 billion therapeutic microparticles per milliliter, or about 1.4 billion therapeutic microparticles per milliliter to about 2.2 billion therapeutic microparticles per milliliter of formulation. In certain embodiments, the formulation contains about 500 million therapeutic microparticles per milliliter of formulation to about 1.5 billion therapeutic microparticles per milliliter of formulation. [Example]

[0098] The present disclosure will now be described using examples. These examples are intended to illustrate the function of the present disclosure and are not intended to limit the scope of the present disclosure. 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 present disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the methods and compositions of the present disclosure, the present specification describes exemplary methods, devices, and materials. It is understood that the present disclosure is not limited to the specific methods and experimental conditions described, and that such methods and conditions may vary widely.

[0099] therapeutic microparticles Synthesis of pDox Generally, doxorubicin-conjugated poly(L-glutamic acid) polymers (pDox) can be prepared according to the method described in (Xu et al., “An injectable nanoparticle generator enhances delivery of cancer therapeutics,” Nature Biotechnology, 34, 414-418 (2016)).

[0100] For example, a hydrazide group can be conjugated to the glutamic acid side chain of poly(L-glutamic acid) via an acid anhydride reaction for the synthesis of pDox. N-morpholino is added to anhydrous dimethylformamide (DMF) containing poly(L-glutamic acid), followed by dropwise addition of isobutyl chloroformate at 4°C under argon gas. After stirring for 15 minutes, tert-butyl carbazate in DMF is added. The resulting solution is reacted at 4°C for 30 minutes, followed by 2 hours at 25°C. To obtain poly(L-glutamic acid hydrazide)-poly(L-glutamic acid) copolymer, the resulting product is stirred with TFA (trifluoroacetic acid) at room temperature for 1 hour.

[0101] Approximately 100 mg of poly(L-glutamic acid hydrazide)-poly(L-glutamic acid) copolymer was dissolved in 200 ml of anhydrous methanol or DMSO (dimethyl sulfoxide) and 100 μL of trifluoroacetic acid was added. Doxorubicin hydrochloride was then added, and the mixture was stirred under argon gas at 25°C for 48 hours. pDox was concentrated, dialyzed in methanol, and purified with SEPHADEX®-LH20 (AMERSHAM®, Pharmacia Biotech Co.).

[0102] Alternatively, conjugation of tert-butyl carbazate with poly(L-glutamic acid) can be prepared using HBTU / HOBt as coupling reagents rather than a mixed anhydride coupling reaction. For example, 2 grams of poly-L-glutamic acid is added to 100 mL of anhydrous DMF (dimethylformamide) in a 250 mL flask, heated and stirred until a clear solution forms, and then cooled to room temperature. 960 mg of HBTU (N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl) uronium hexafluorophosphate, CAS 94790-37-1), 380 mg of HOBt (hydroxybenzotriazole, CAS 123333-53-9), 336 mg of tert-butyl carbazate (CAS 870-46-2), and 800 μL of N,N-diisopropylethylamine are added sequentially. The reaction is stirred under argon at room temperature for 24 hours.

[0103] Synthesis of porous silicon particles The synthesis of porous silicon microparticles (injectable nanoparticle generators, iNPGs) and their chemical surface modification are described herein. The porous silicon microparticles are synthesized according to the method described in (Xu et al., “An injectable nanoparticle generator enhances delivery of cancer therapeutics,” Nature Biotechnology, 34, 414-418 (2016)).

[0104] For example, a heavily doped p++ type (100) silicon wafer (Silicon Quest, Inc., Santa Clara, CA) with a resistivity of 0.005 ohm-cm is used as the silicon substrate. A 400 nm porous layer is deposited using a 1:3 HF (49%):ethanol solution at 7 mA / cm. -1 A current of 0.05 V was applied for 125 minutes to form the porous bilayer. The current was then increased to 76 mA / cm for 8 minutes, forming a highly porous emissive layer. Low-pressure chemical vapor deposition at 400°C deposited a 40 nm SiO2 layer. Standard photolithography was used to pattern a 2.6 μm circular pattern with a 3.4 μm pitch across the SiO2-encapsulated porous layer using a contact aligner (KARL SUSS® MA6 Mask Aligner) and NR9-500P photoresist (FUTURREX® Franklin, NJ, USA). The pattern was transferred to the porous bilayer by dry etching with CF4 plasma (PLASMATHERM® 790, 25 sccm CF4, 100 mTorr, 200 W RF). The encapsulating SiO2 layer was removed with 49% HF and sonicated in isopropanol to release the microparticles from the substrate. The particles were treated with H2O2 at 100°C. 、 The surface was then oxidized. The microparticles were then modified with 2% (v / v) 3-aminopropyltriethoxysilane (APTES) in isopropanol at 55°C for 48 hours to conjugate primary amines on the microparticle surface, yielding APTES-modified porous silicon microparticles. The microparticles were found to have a size of approximately 2.6 μm, a thickness of approximately 700 nm, and pore diameters of 40 nm to 80 nm.

[0105] Loading of pDox onto microparticles (iNPG) to prepare pDox-iNPG iNPG-pDox microparticles are constructed by loading concentrated pDox molecules (>10 mg / ml in methanol) onto APTES-modified porous silicon microparticles, followed by vacuum drying. This loading process is repeated until the nanopores are completely filled with pDox. Preparing therapeutic microparticles—iNPG-pDox—poses challenges. For example, iNPG-pDox consists of disk-shaped porous silicon microparticles (iNPG particles) with a diameter of 2–3 micrometers and poly(L-glutamic acid) polymers (pDox) conjugated with doxorubicin. pDox is loaded into the nanopores of the iNPG formulation to obtain therapeutic microparticles (iNPG-pDox). However, iNPG particles tend to form aggregates in water or in many organic solvents, such as dimethylformamide (DMF), which is one of the best solvents for pDox. When pDox is loaded into iNPG microparticles in DMF, some small and large aggregates are formed. This tends to limit the amount of pDox microparticles that can be loaded into an iNPG formulation without incurring any aggregation.

[0106] A series of experiments were performed to determine (i) a solvent for dissolving pDox and (ii) an acceptable pDox-to-iNPG ratio to avoid any aggregation or precipitation. Methanol was found to be a suitable organic solvent for dissolving pDox and suspending iNPG microparticles. Additionally, an acceptable pDox-to-iNPG ratio was found to be less than 1.6 mg pDox / billion iNPG. A pDox-to-iNPG ratio greater than 1.6 mg pDox / billion iNPG resulted in particle precipitation and the formation of high aggregates.

[0107] formulation Various formulations were prepared using varying amounts of sodium carboxymethylcellulose, mannitol, and poloxamer 188. The experimental protocol details, along with the amounts of each ingredient, are provided in Table 1 below. [Table 1]

[0108] In Table 1, "-1" indicates that the formulation contains 1.7 wt. % of component A (CMC-Na) based on the total weight of the formulation. Similarly, "0" indicates that the formulation contains 1.2 wt. % of CMC-Na, and "1" indicates that the formulation contains 0.7 wt. % of CMC-Na, based on the total weight of the formulation. Each formulation has approximately 2 billion therapeutic microparticles per mL.

[0109] All ingredients in the preparation of the formulation are accurately weighed according to Table 1 set forth hereinabove, added in different combinations (according to Table 2 set forth hereinbelow) into water and mixed thoroughly to obtain a formulation for intravenous injection.

[0110] Therapeutic microparticles-iNPG-pDox (as prepared in the examples above) are added to prepare formulations so that each formulation has approximately 2 billion therapeutic microparticles per mL of formulation. Each formulation is then tested for suspension of microparticles.

[0111] Table 2 below shows the effect of varying the amount of each component of the formulation on the suspension time of the iNPG-pDox microparticles in the formulation, ie, the duration that the iNPG-pDox microparticles remain suspended within the formulation. [Table 2]

[0112] As can be seen from Table 2, iNPG-pDox (therapeutic microparticles) remain uniformly suspended for approximately 105 minutes in a formulation containing 1.2% CMC-Na, 0.2% poloxamer 188, and 0.5% mannitol.

[0113] To evaluate the effect of different concentrations of iNPG-pDox on the suspension time of iNPG-pDox microparticles, formulations were prepared using 1.2% CMC-Na, 0.2% poloxamer 188, and 0.5% mannitol containing different amounts of iNPG-pDox microparticles. The test results are shown in Table 3 herein below. [Table 3]

[0114] In Table 3, an "X" indicates that the particles settled, and a "check" indicates that the particles remained suspended. As can be seen from Table 3, even when the formulation contained approximately 2 billion iNPG-pDox therapeutic microparticles, the formulation was able to maintain the iNPG-pDox therapeutic microparticles in suspension for approximately 2 hours. When the formulation contained a lower concentration of iNPG-pDox therapeutic microparticles (0.5 billion), the formulation was able to maintain the iNPG-pDox therapeutic microparticles in suspension for approximately 3 hours.

[0115] Effect of rotational flow on suspension time of iNPG-pDox microparticles Is it possible to keep therapeutic microparticles in suspension for more than 3 hours?

[0116] To further understand the effect of agitation or rotational flow on the suspension time of iNPG-pDox microparticles in a formulation, the formulations described above (having 0.5-3 billion iNPG-pDox microparticles) were rotationally flowed at 25 RPM. The results are shown in Figure 1. As can be seen from Figures 1A-1D, maintaining the formulation on the rotor can improve suspension time for formulations containing higher concentrations of iNPG-pDox therapeutic microparticles (i.e., 3 billion particles / mL formulations), with the microparticles remaining fully suspended for approximately 2.5 hours.

[0117] Comparison of suspension time of therapeutic microparticles (MSV / siRNA) in this formulation and water Do the formulations herein only work with pDox-loaded therapeutic microparticles, or do the formulations suspend other therapeutic microparticles with different active agents?

[0118] A different active agent, MSV / siRNA (multistage vectorized siRNA), was suspended using a formulation containing 1.2% CMC-Na, 0.2% poloxamer 188, and 0.5% mannitol. Figure 2 shows a comparison of the suspension time of therapeutic microparticles (MSV / siRNA) in this formulation with that in water, a control. As can be seen from Figure 2, the MSV / siRNA microparticles maintained the suspension of the formulation for up to 3 hours, whereas in water, the MSV / siRNA microparticles settled within 30 minutes.

[0119] The studies described above demonstrate that the formulations of the present disclosure provide a suspension of therapeutic microparticles in the formulation long enough for administration to subjects requiring intravenous injection.

[0120] Without further elaboration, it is believed that one skilled in the art can, in light of the description herein, utilize the present invention to its fullest extent. The embodiments described herein are to be construed as illustrative, and not limitative of the remainder of the disclosure in any way. While some embodiments of the present invention have been shown and described, numerous changes and modifications thereof will occur to those skilled in the art.

Claims

1. A formulation for intravenous injection, comprising: About 0.80 weight percent to about 1.60 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation; about 0.12 weight percent to about 0.28 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), based on the total weight of the formulation; about 0.22 weight percent to about 0.78 weight percent mannitol, based on the total weight of the formulation; and The formulation comprises water.

2. A therapeutic microparticle, said therapeutic microparticle comprising an active agent, a polymer-active agent conjugate, or a combination thereof; 10. The formulation of claim 1, wherein the polymer-active agent conjugate further comprises a therapeutic particle comprising a polymeric carrier conjugated to the active agent.

3. the formulation comprises about 1.19 weight percent to about 1.54 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation; or 10. The formulation of claim 1, wherein the formulation comprises about 1.33 weight percent to about 1.47 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation.

4. the formulation comprises about 0.19 weight percent to about 0.21 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), based on the total weight of the formulation; or the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) has a weight average molecular weight of about 8,400 daltons; or The poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) is represented by Formula I 【Chemical 1】 2. The formulation of claim 1, having the structure: wherein X is 72-88, Y is 23-30, and Z is 72-88.

5. 10. The formulation of claim 1, wherein the formulation contains from about 0.48 weight percent to about 0.66 weight percent mannitol based on the total weight of the formulation, or from about 0.49 weight percent to about 0.63 weight percent mannitol based on the total weight of the formulation.

6. the formulation comprises from about 500 million therapeutic microparticles per milliliter of the formulation to about 3 billion therapeutic microparticles per milliliter of the formulation, or the formulation comprises from about 1 billion therapeutic microparticles per milliliter of the formulation to about 2.5 billion therapeutic microparticles per milliliter of the formulation, or the therapeutic microparticles have a longest dimension of about 1,700 nm to about 3,000 nm, a smallest dimension of about 100 nm to about 200 nm, or a combination thereof; or 3. The formulation of claim 2, wherein the therapeutic microparticles have a disc shape with a diameter of about 900 nm to about 2,800 nm and a thickness of about 200 nm to about 800 nm.

7. The polymeric carrier may be selected from poly-L-glutamic acid, poly(lactic acid), poly(glycolic acid), poly(D-lactic acid-co-glycolic acid), poly(L-lactic acid-co-glycolic acid), poly(D,L-lactic acid-co-glycolic acid), poly(caprolactone), poly(valerolactone), poly(hydroxybutyric acid), poly(hydrovaleric acid), polydioxynanone, and combinations thereof; The active carrier may be a drug, a bioactive compound, a chemotherapeutic agent, an immunosuppressant, a cytokine, a cytotoxic agent, a nucleic acid degrading compound, an imaging agent, a prodrug enzyme, a gene, a nucleic acid, shRNA, siRNA, a DNA fragment, an RNA fragment, a plasmid, or any combination thereof; 3. The formulation of claim 2, wherein the therapeutic microparticles are porous silicon microparticles containing a polymer of L-glutamic acid conjugated with doxorubicin, and the polymer has a weight average molecular weight of 70-80 kDa.

8. 1. A method for preparing a formulation for intravenous injection, comprising: forming a formulation by adding sodium carboxymethylcellulose, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), and mannitol to water, either simultaneously or in any order; the formulation comprising about 0.80 weight percent to about 1.60 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation; the formulation comprises about 0.12 weight percent to about 0.28 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), based on the total weight of the formulation; The method, wherein the formulation comprises about 0.22 weight percent to about 0.78 weight percent mannitol, based on the total weight of the formulation.

9. adding therapeutic microparticles to the formulation to form a pharmaceutical formulation, wherein the therapeutic microparticles comprise porous silicon microparticles containing polymeric doxorubicin or porous silicon microparticles containing siRNA; or adding therapeutic microparticles to the formulation to form a pharmaceutical formulation, wherein the therapeutic microparticles comprise porous silicon microparticles comprising an active agent, a polymer-active agent conjugate, or a combination thereof, and the polymer-active agent conjugate comprises a polymeric carrier conjugated to the active agent; or 9. The method of claim 8, further comprising adding therapeutic microparticles to the formulation to form a pharmaceutical formulation, wherein the therapeutic microparticles are porous silicon microparticles comprising a polymer of L-glutamic acid conjugated to doxorubicin, the polymer having a weight average molecular weight of 70-80 kDa.

10. the polymeric carrier comprises poly-L-glutamic acid, poly(lactic acid), poly(glycolic acid), poly(D-lactic acid-co-glycolic acid), poly(L-lactic acid-co-glycolic acid), poly(D,L-lactic acid-co-glycolic acid), poly(caprolactone), poly(valerolactone), poly(hydroxybutyric acid), poly(hydrovaleric acid), polydioxynanone, or a combination thereof; or 10. The method of claim 9, wherein the active agent comprises a drug, a bioactive compound, a chemotherapeutic agent, an immunosuppressant, a cytokine, a cytotoxic agent, a nucleic acid degrading compound, an imaging agent, a prodrug enzyme, a gene, a nucleic acid, shRNA, siRNA, a DNA fragment, an RNA fragment, a plasmid, or any combination thereof.

11. 10. The method of claim 8, further comprising adding therapeutic microparticles to the formulation before, during, or after adding the sodium carboxymethylcellulose, the poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), or the mannitol to water to form a pharmaceutical formulation.

12. adding therapeutic microparticles to said formulation to form a pharmaceutical formulation, said therapeutic microparticles being said porous silicon microparticles comprising said polymer of L-glutamic acid conjugated with doxorubicin, prior to said forming; forming a suspension of said therapeutic microparticles, said forming a suspension of said therapeutic microparticles comprising: dissolving a poly(L-glutamic acid) polymer conjugated to doxorubicin in a liquid to form a doxorubicin solution, the liquid comprising about 50 weight percent to 100 weight percent methanol, based on the total weight of the doxorubicin solution; and 10. The method of claim 9, further comprising contacting porous silicon microparticles with the doxorubicin solution to form therapeutic microparticles.

13. After forming the suspension of therapeutic particles and prior to forming the pharmaceutical formulation, Centrifuging the doxorubicin solution to separate a supernatant, and separating the supernatant from an aliquot of the doxorubicin solution; or after forming the suspension of therapeutic particles and before forming the pharmaceutical formulation; and 13. The method of claim 12, further comprising applying vibration, ultrasound, or a combination thereof to agitate the doxorubicin solution.

14. 1. A method of administering therapeutic microparticles to a subject in need thereof, comprising:

1. A pharmaceutical formulation comprising: About 0.80 weight percent to about 1.60 weight percent sodium carboxymethylcellulose, based on the total weight of the formulation; about 0.12 weight percent to about 0.28 weight percent poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), based on the total weight of the formulation; about 0.22 weight percent to about 0.78 weight percent mannitol, based on the total weight of the formulation; Therapeutic microparticles, and providing said solution containing water; adding said therapeutic formulation to an intravenous line; and connecting the intravenous line to the subject.

15. the therapeutic microparticles comprise an active agent, a polymer-active agent conjugate, or a combination thereof; The polymer-active agent conjugate comprises a polymeric carrier conjugated to the active agent; or 15. The method of claim 14, wherein the pharmaceutical formulation comprises from about 500 million therapeutic microparticles per milliliter to about 3 billion therapeutic microparticles per milliliter of the pharmaceutical formulation.

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