CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application No. 62/832,770, filed on April 11, 2019, which is hereby incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant R01CA164789, R01EB020147, R01CA197111, R01CA220841, and R01CA204968 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention is generally in the field of drug delivery, and in particular, a method of delivering drugs to the brain using coated particles to penetrate brain tissue.

BACKGROUND OF THE INVENTION

While the blood brain barrier has long been considered the crucial interface for therapeutic efficacy within the CNS, more recently poor distribution of agents within the brain itself has emerged as a major delivery challenge. See Arifin, et al. Pharm Res 26, 2289 (2009), Bobo el al., Proc Natl Acad Sci U S A 91, 2076 (1994), Kunwar et al. , Neuro Oncol 12, 871 (2010), Kunwar et al. , J Clin Oncol 25, 837 (2007), Rosso et al. , Cancer Res 69, 120 (2009), and Sampson et al, J Neurosurg 113, 301 (2010).

The extracellular space (ECS) in the brain represents the major pathway for movement of many signaling molecules and metabolites as well as therapeutic and diagnostic substances. This space between cells comprises 15-20% of the total brain volume, contains charged and hydrophobic regions, and shifts with changes in cerebral metabolic activity, blood flow, and spinal fluid dynamics. Importantly, the ECS may be more complex in certain pathogenic states, such as intrinsic invasive brain tumors. Achieving widespread distribution in the brain parenchyma is essential when it comes to the design of therapeutic nanoparticles (NPs) for treating brain diseases (1, 2). NPs delivery systems used for the treatment of brain tumors and other CNS diseases is challenging due to the presence of multiple anatomical and physiological barriers (e.g. blood-brain barrier, stability in blood circulation, brain tissue barrier, drug clearance form cerebrospinal fluid, high vascularity, and tissue heterogeneity). Furthermore, the lack of efficacy of locally delivered systems stem from the impaired diffusional capabilities of the chemotherapeutic released after infusion. This limits their application for long-term treatment regimens, further supporting a need for a NP platform that can penetrate within the brain and provide sustained release of a therapeutic.

It is therefore an object of the present invention to provide the particle characteristics that enable maximum drug loading and release times for delivery of therapeutic, prophylactic and diagnostic agents to the brain, while optimizing or maximizing penetration.

It is a further object of the present invention to provide methods to assist the delivery of these particles to further enhance the penetration and delivery of the cargo of these particles to the brain.

SUMMARY OF THE INVENTION

Dosage formulations containing nanoparticles, particularly nanoparticles which exhibit increased rates of diffusion through the brain parenchyma, and methods of making and using thereof, are described herein. In a particular embodiment, the nanoparticles have encapsulated therein and/or associated with the surface of the particles, one or more therapeutic, prophylactic, and/or diagnostic agents.

Generally, the nanoparticles are made up entirely of Generally Recognized As Safe (GRAS) materials, in compliance with the regulations provided by the U.S. Food and Drug Administration in sections 20!(s) and 409 of the Federal Food, Drug, and Cosmetic Act and 21 C.F.R. 170 and 181-186. In one embodiment, the core is made up with one or more hydrophobic polymers, preferably poly(lactic acid-co-glycolic acid) (PLGA)

45355640 or other biodegradable, biocompatible polymer such other poly(hydroxy acids), poly anhydrides, and polyhydroxybutyrates such as PHB3 and PHB4. In a particular embodiment, the particles are coated with one or more materials which facilitate or enhance diffusion of the particles, particularly through the brain. The coating material can be a surfactant or a hydrophilic material, such as a hydrophilic polymer. In one embodiment, the material is a triblock copolymers of polyethylene oxide-polypropylene oxide- polyethylene oxide (PEO-PPO-PEO), available commercially as a

PLURONIC ^® or POLOXAMER ^® . In one embodiment, the particles are densely coated with the coating material. In a particular embodiment, the density of the coating is such that the surface of the particle is near-neutral as measured by the zeta potential of the surface and/or the particles exhibit significantly reduced adhesion to tissue compared to uncoated particles. In another embodiment, the density of the coating is such that the particle penetrates brain tissue at a rate significantly faster than an uncoated particle. The density of the coating can be expressed as units per nm ² or as the ratio of the mass of the coating to the mass of the particle or as the weight percent of the coating.

In one embodiment, the particles have an average size (e.g., diameter) from about 60 to about 230 nm, between 80 and 230 nm, preferably from about 80 to about 150 nm, or more preferably between about 150 and about 230 nm. In another embodiment, the particles have an average size (e.g., diameter) from about 150 and about 230 nm, such as 150, 160, 170, 180,

190, 200, 210, 220 nm.

The nanoparticles can be combined with one or more

pharmaceutically acceptable excipients and/or carriers to form

pharmaceutical formulations. The formulations can be administered directly or indirectly to the brain using methods of administration known in the art.

In preferred embodiment, the nanoparticles are directly injected to the brain via intracranial administration by convection enhanced delivery (“CED”).

The nanoparticles are formulated to release an effective amount of the therapeutic, prophylactic, or diagnostic agent over a period of at least 10

45355640 minutes, 20 minutes, 30 minutes, one hour, two hours, hour hours, six hours, ten hours, one day, three days, seven days, ten days, two weeks, one month, or longer.

The nanoparticles can be formulated with an osmolality between about 300 mOsm/kg and about 1000 mOsm/kg, preferably between about 400 mOsm/kg and about 800 mOsm/kg, effective to increase volumetric distribution of the nanoparticles at the site of administration in the brain relative to an equivalent formulation with an osmolality of less than 300 mOsm/kg. Statistically significant improvement of therapeutic distribution enabled by these non-adhesive, brain-permeable nanoparticles and osmosis- driven brain extracellular matrix (ECM) modulation will significantly enhance drug and gene delivery within the CNS, offering higher drug payload, improved drug loading efficiency, and significantly longer drug release durations.

As demonstrated by the examples, nanoparticles (NPs) have been developedthat provide widespread and sustainable delivery of small- molecule drugs to the brain solely based on generally recognized as safe (GRAS) materials, including poly (lactic-co-glycolic acid) (PLGA) and triblock copolymers of polyethylene oxide -polypropylene oxide- polyethylene oxide (PEO-PPO-PEO), also used as surfactants, which provides a clear regulatory path for the safety assessment in the clinic. A family of PEO-PPO-PEO surfactant polymers, Pluronic F68, F98, and F127, possessing a range of relative molecular weights of PEO and PPO was tested and a lead polymer that provides most stably surface non-adhesive coatings for efficient particle penetration through the brain tissue was demonstrated. NPs as large as -200 nm in diameters can efficiently penetrate the brain parenchyma when the NPs surfaces are coated with the lead PEO-PPO-PEO, Pluronic F127. This is of importance as the largest NPs previously shown to efficiently penetrate the brain tissues possessed diameters of -110 nm and larger NPs allows delivery of a wider array of drugs to the brain at higher drug concentrations.

45355640 The examples also demonstrate the use of a simple osmotic method to non-invasively and temporarily increase the pore sizes of brain ECM, thereby further enhancing distribution of therapeutic NPs in the brain.

Synergistic improvement of therapeutic distribution enabled by non adhesive, brain-permeable NPs and osmosis-driven ECM modulation will likely be a game-changer for brain therapy given that most of the neurological disorders, including malignant gliomas, are characterized by widespread disease areas within the brain

The ability to achieve brain penetration with larger particles will significantly improve drug and gene delivery within the CNS since larger particles offer higher drug payload, improved drug loading efficiency, and significantly longer drug release durations.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1B are bar graphs showing (FIG.1A) hydrodynamic diameters of 30 mg/ml PLGA NPs measured by DLS in 10 mM NaCI at pH 7.0. Mean ± SEM (n > 3) and (FIG. IB) z- potential of uncoated NPs or coated NPs with Pluronic solutions, F68, F98, and F127 measured by laser doppler anemometry in 10 mM NaCI at pH 7.0.. Mean ± SEM (n > 3). Figure 1C is a line graph showing colloidal stability (hydrodynamic diameter, nm) of uncoated NPs or coated NPs with Pluronic solutions, F68, F98, and F127 over a period of 12 hours in artificial cerebrospinal fluid (aCSF) at 37°C.

Figures 2A and 2B are bar graphs showing volume of distribution obtained by confocal microscopy immediately after NPs treatment mediated by CED of uncoated NPs or coated NPs with Pluronic solutions, F68, F98, and F127 administered into the rats striatum via CED in healthy rat brains immediately after NPs-treatment. n > 4 rats. Differences are statistically significant as indicated (*p < 0.05, **p < 0.01 ***P< 0.001) (Figure 2A) and showing quantification of the volume of distribution of PLGA NPs (mm ³ ) (n > 4 rats). Differences are statistically significant as indicated (*p < 0.05, **p < 0.01 ***p < 0.001) (Figure 2B).

45355640 Figure 3 is a graph of the hydrodynamic diameter (nm) over time (hours) for different concentrations of NPs: 5 (closed circle), 10 (open circle), 30 (closed square) and 50 (open square).

Figure 4 is a bar graph showing volumetric distribution of

F127/PLGA NPs of different hydrodynamic diameters at 60 nm, 130 nm,

170 nm, and 200 nm administered via CED in healthy rat brains n > 4 rats. Differences are statistically significant as indicated (t-test *p < 0.05)

Figure 5 is a bar graph of the histopathological analysis and safety profile, the graph showing scores of inflammation and hemorrhage 3 days following administration of medical-grade normal saline (NS), F98/PLGA NPs, and F127/PLGA NPs (0: no inflammation/hemorrhage; 1: mild; 2: moderate; 3: severe) of F98/PLGA NPs and F127/PLGA NPs following CED administration. Representative images of the injection site with hematoxylin and eosin staining in healthy brain tissue 3 days after CED at 20X (top, scale bar = 500 pm) and 200X (bottom, scale bar = 100 pm) magnitude. Normal saline was used as a control. Inflammation and hemorrhage were scored by a board-certified neuropathologist using a custom scale (0: no

inflammation/hemorrhage, 1: mild, 2: moderate, 3: severe).

Figures 6 A and 6B are representative 3D-rendered images depicting orthotopic F98-mKate brain tumors (dark gray) and the volumetric distribution of F127/PLGA NPs (light blue, outlined by blue-dashed lines) in the tumor bulk, obtained by stacking multiple sequential confocal images. Fig. 6 A shows tumor bulk in coronal and sagittal views. Fig. 6B shows areas where F127/PLGA NPs are surrounding the tumor in coronal and sagittal views. Scale bar = 2 mm. 3D-rendered representation depicting orthotopic F98-mKate brain tumors and the volumetric distribution of F127/PLGA NPs (grey outline) in the tumor bulk, obtained by stacking multiple sequential confocal images.

Figure 7 is a bar graph showing volumetric distribution of

F127/PLGA NPs at the infusion sites of fresh F127/PLGA NPs or lyophilized F127/PLGA NPs at various osmotic pressures, 480 mOsm/kg, 370 mOsm/kg, and 300 mOsm/kg immediately after NPs-treatment

45355640 administered via CED in healthy rat brains, obtained by confocal microscopy immediately after NPs-treatment. Sale bar = 1 mm. (n > 3 rats). Differences are statistically significant as indicated (*p < 0.05, **p < 0.01).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The term "biocompatible" as used herein refers to one or more materials that are neither themselves toxic to the host (e.g., an animal or human), nor degrade (if the material degrades) at a rate that produces monomeric or oligomeric subunits or other byproducts at toxic

concentrations in the host.

The term "biodegradable" as used herein means that the materials degrades or breaks down into its component subunits, or digestion, e.g., by a biochemical process, of the material into smaller (e.g., non-polymeric) subunits.

The term "corresponding particle" or“reference particle” as used herein refers to a particle that is substantially identical to another particle to which it is compared, but typically lacking a surface modification to promote transport differences through the pores in the ECS of the brain. A corresponding particle may be of similar material, density, and size as the particle to which it is compared. In certain embodiments, a corresponding particle is a carboxyl-modified polystyrene (PS) particle, e.g., available from Molecular Probes, Eugene, OR. In certain embodiments, a comparable particle is a polystyrene particle that has carboxyl, amine or sulfate aldehyde surface modifications.

The term "DNA" refers to a polymer of deoxynucleotides. Examples of DNA include plasmids, gene therapy vector, and a vector designed to induce RNAi.

The term "diameter" is art-recognized and is used herein to refer to either of the physical diameter or the hydrodynamic diameter. The diameter of an essentially spherical particle may refer to the physical or hydrodynamic diameter. The diameter of a nonspherical particle may refer preferentially to the hydrodynamic diameter. As used herein, the diameter of a non-spherical

45355640 particle may refer to the largest linear distance between two points on the surface of the particle. When referring to multiple particles, the diameter of the particles typically refers to the average diameter of the particles. Particle diameter can be measured using a variety of techniques in the art including, but not limited to, dynamic light scattering.

“Sustained release" as used herein refers to release of a substance over an extended period of time in contrast to a bolus type administration in which the entire amount of the substance is made biologically available at one time.

The term "microspheres" is art-recognized, and includes substantially spherical colloidal structures, e.g., formed from biocompatible polymers such as subject compositions, having a size ranging from about one or greater up to about 1000 microns. In general, "microcapsules," also an art- recognized term, may be distinguished from microspheres, because microcapsules are generally covered by a substance of some type, such as a polymeric formulation. The term "microparticles" is also art-recognized, and includes microspheres and microcapsules, as well as structures that may not be readily placed into either of the above two categories, all with dimensions on average of less than about 1000 microns. A microparticle may be spherical or nonspherical and may have any regular or irregular shape. If the structures are less than about one micron in diameter, then the corresponding art-recognized terms "nanosphere," "nanocapsule," and "nanoparticle" may be utilized. In certain embodiments, the nanospheres, nanocapsules and nanoparticles have an average diameter of about 500 nm, 200 nm, 100 nm,

50 nm, 10 nm, or 1 nm.

A composition containing microparticles or nanoparticles may include particles of a range of particle sizes. In certain embodiments, the particle size distribution may be uniform, e.g., within less than about a 20% standard deviation of the mean volume diameter, and in other embodiments, still more uniform, e.g., within about 10% of the median volume diameter.

The term "particle" as used herein refers to any particle formed of, having attached thereon or thereto, or incorporating a therapeutic, diagnostic

45355640 or prophylactic agent, optionally including one or more polymers, liposomes micelles, or other structural material. A particle may be spherical or nonspherical. A particle may be used, for example, for diagnosing a disease or condition, treating a disease or condition, or preventing a disease or condition.

The phrases "parenteral administration" and "administered parenterally" are art-recognized terms, and include modes of administration other than enteral and topical administration, such as injections, and include without limitation intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradennal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

The term "surfactant" as used herein refers to an agent that lowers the surface tension of a liquid.

The term "therapeutic agent" refers to an agent that can be administered to prevent or treat a disease or disorder. Examples include, but are not limited to, a nucleic acid, a nucleic acid analog, a small molecule, a peptidomimetic, a protein, peptide, carbohydrate or sugar, lipid, or surfactant, or a combination thereof.

The term "treating" preventing a disease, disorder or condition from occurring in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain.

The term "targeting moiety" as used herein refers to a moiety that localizes to or away from a specific locale. The moiety may be, for example,

45355640 a protein, nucleic acid, nucleic acid analog, carbohydrate, or small molecule. Said entity may be, for example, a therapeutic compound such as a small molecule, or a diagnostic entity such as a detectable label. Said locale may be a tissue, a particular cell type, or a subcellular compartment. In one embodiment, the targeting moiety directs the localization of an active entity. The active entity may be a small molecule, protein, polymer, or metal. The active entity may be useful for therapeutic, prophylactic, or diagnostic purposes.

The phrase "pharmaceutically acceptable" refers to compositions, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, solvent or encapsulating material involved in carrying or transporting any subject composition, from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient.

The term "pharmaceutically acceptable salts" is art-recognized, and includes relatively non-toxic, inorganic and organic acid addition salts of compounds. Examples of pharmaceutically acceptable salts include those derived from mineral acids, such as hydrochloric acid and sulfuric acid, and those derived from organic acids, such as ethanesulfonic acid,

benzenesulfonic acid, and p-toluenesulfonic acid. Examples of suitable inorganic bases for the formation of salts include the hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, potassium, calcium, magnesium, aluminum, and zinc. Salts may also be formed with suitable organic bases, including those that are non-toxic and strong enough to form such salts. For purposes of illustration, the class of such organic bases may

45355640 include mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and triethylamine; mono-, di- or trihydroxyalkylamines such as mono-, di-, and triethanolamine; amino acids, such as arginine and lysine; guanidine; N- methylglucos amine; N-methylglucamine; L-glutamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine;

The term "prolonged residence time" as used herein refers to an increase in the time required for an agent to be cleared from a patient's body, or organ or tissue of that patient. In certain embodiments, "prolonged residence time" refers to an agent that is cleared with a half-life that is 10%, 20%, 50% or 75% longer than a standard of comparison such as a comparable agent without a mucus-resistant coating. In certain embodiments, "prolonged residence time" refers to an agent that is cleared with a half-life of 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, or 10000 times longer than a standard of comparison such as a comparable agent without a coating the promotes diffusion through the pores of the ECS of the brain.

The term "therapeutically effective amount" refers to an amount of the therapeutic agent that, when incorporated into and/or onto particles described herein, produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. The effective amount may vary depending on such factors as the disease or condition being treated, the particular targeted constructs being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation. In some embodiments, the term “effective amount” refers to an amount of a therapeutic agent or prophylactic agent to reduce or diminish the symptoms of one or more diseases or disorders of the brain, such as reducing tumor size (e.g., tumor volume) or reducing or diminishing one or more symptoms of a neurological disorder, such as memory or learning deficit, tremors or shakes, etc. In still other embodiments, an“effective amount” refers to the amount of a therapeutic agent necessary to repair damaged neurons and/or induce regeneration of neurons.

45355640 The terms "incorporated" and "encapsulated" refers to incorporating, formulating, or otherwise including an active agent into and/or onto a composition that allows for release, such as sustained release, of such agent in the desired application. The terms contemplate any manner by which a therapeutic agent or other material is incorporated into a polymer matrix, including for example: attached to a monomer of such polymer (by covalent, ionic, or other binding interaction), physical admixture, enveloping the agent in a coating layer of polymer, and having such monomer be part of the polymerization to give a polymeric formulation, distributed throughout the polymeric matrix, appended to the surface of the polymeric matrix (by covalent or other binding interactions), encapsulated inside the polymeric matrix, etc. The term "co-incorporation" or "co-encapsulation" refers to-the incorporation of a therapeutic agent or other material and at least one other therapeutic agent or other material in a subject composition.

More specifically, the physical form in which any therapeutic agent or other material is encapsulated in polymers may vary with the particular embodiment. For example, a therapeutic agent or other material may be first encapsulated in a microsphere and then combined with the polymer in such a way that at least a portion of the microsphere structure is maintained.

Alternatively, a therapeutic agent or other material may be sufficiently immiscible in the polymer that it is dispersed as small droplets, rather than being dissolved, in the polymer.

II. Compositions

Non-adhesive, brain-permeable particles suitable for delivering one or more active agent, particularly one or more active agents to prevent, treat or diagnose one or more diseases or disorders of the brain are described. These non-adhesive, brain-permeable nanoparticles with a diameter as large as 200nm can diffuse rapidly in the brain ECS, preferably having neutral surface charge. In preferred embodiments, these particles are made up entirely of Generally Recognized As Safe (GRAS) materials. Synthetic particles composed entirely of GRAS materials will facilitate rapid translation of nanomaterials-based products into humans for the treatment of

45355640 numerous diseases and conditions that affect the brain. As discussed in the examples below, exemplary particles include poly(lactic-co-glycolic acid) (PLGA) particles coated with triblock copolymers of polyethylene oxide- polypropylene oxide-polyethylene oxide (PEO-PPO-PEO).

A. Particles

1. Size

The ability of larger particles to diffuse into tissues holds many important implications for nanoparticle -based drug delivery and diagnostic systems. First, large particles have an enormous advantage over smaller particles for delivering drugs or diagnostic agents to tissues. This is not only because theoretical drug payload per particle increases with particle radius to the third power, but also because the ability to encapsulate a wide variety of therapeutics is significantly improved with the greater particle volume. By increasing particle size from 30 nm (expected to be capable of diffusing within ECS) to 100 nm (not previously expected to be capable), one can achieve more than 1000-fold higher drug loading per particle. Another advantage is that it is very difficult to attain slow release kinetics of molecules entrapped in small particles, whereas it is relatively

straightforward to do so with larger particles. Taken together, these results show that the increased particle size described here should have significant impact on the ability to use nano-sized carriers for delivery of diagnostic and therapeutic agents in the brain.

In some embodiments, the particles have an average diameter greater than the pores in the extracellular space (ECS) of the brain. The brain ECS was found to have pores as large as 300 nm, with a high percentage having pores larger than 100 nm. In particular embodiments, the particles have an average diameter up 230 nm, preferably from about 60 to about 200 nm, more preferably from about 110 to about 200 nm as measured using dynamic light scattering. In another embodiment, the particles have a diameter from about 110 to about 230 nm, such as about 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, or 220 nm. The ranges above are inclusive of all values between the minimum and maximum values.

45355640 In another embodiment, the particles have an average diameter such that a majority of the particles do not become localized within cells or microdomains within tissue compared to larger particles. In particular embodiment, the particles have a diameter greater than 60 nm, particularly greater than 80 nm or greater than 100 nm and preferably less than 250 nm, more preferably less than 230 nm. The particles may have a size less than 60 nm provided the coating(s) resulting in enhanced penetration of a higher percentage of the particles.

2. Core polymer

Any number of biocompatible polymers can be used to prepare the nanoparticles. In one embodiment, the biocompatible polymer(s) is biodegradable. In some embodiments, the core polymer is a hydrophobic polymer. Exemplary polymers include, but are not limited to, polyhydroxy acids and copolymers thereof such as poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co- caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), poly anhydrides, poly orthoesters,

The carboxyl termini of carboxylic acid contain polymers, such as lactide- and glycolide-containing polymers, may optionally be capped, e.g., by esterification, and the hydroxyl termini may optionally be capped, e.g. by etherification or esterification. Copolymers of two or more polymers described above, including block and/or random copolymers, may also be employed to make the polymeric particles.

Copolymers of polyethylene glycol (or polyethylene oxide)“PEG” or copolymers or derivatives thereof may be incorporated on or with any of the polymers described above to make the polymeric particles. In certain embodiments, the PEG or derivatives may locate in the interior positions of the copolymer. Alternatively, the PEG or derivatives may locate near or at the terminal positions of the copolymer. In certain embodiments, the microparticles or nanoparticles are formed under conditions that allow

45355640 regions of PEG to phase separate or otherwise locate to the surface of the particles. The surface-localized PEG regions alone may perform the function of, or include, a surface-altering agent.

3. Coatings

The nanoparticles preferably are coated with one or more materials (e.g., surface altering agents) that promote diffusion of the particles through the ECS in the brain by reducing interactions between the particles and brain tissue (e.g., reduce adhesion). Examples of the surface-altering agents include, but are not limited to, anionic proteins (e.g., albumin), surfactants (e.g. polyvinyl alcohol), sugars or sugar derivatives (e.g., cyclodextrin), and polymers.

Certain agents such as cyclodextrin may form inclusion complexes with other molecules and can be used to form attachments to additional moieties and facilitate the functionalization of the particle surface and/or the attached molecules or moieties.

Examples of surfactants include, but are not limited to, L-a- phosphatidylcholine (PC), 1 ,2-dipalmitoylphosphatidycholine (DPPC), oleic acid, sorbitan trioleate, sorbitan mono-oleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, natural lecithin, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, block copolymers of oxyethylene and oxypropylene, synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl monooleate, glyceryl monostearate, glyceryl monoricinoleate, cetyl alcohol, stearyl alcohol, polyethylene glycol 400, cetyl pyridinium chloride, benzalkonium chloride, olive oil, glyceryl monolaurate, corn oil, cotton seed oil, and sunflower seed oil, lecithin, oleic acid, and sorbitan trioleate.

Preferred polymers are generally recognized as safe (GRAS) materials including polyethylene glycol (“PEG”), PEG-containing polymers, and triblock copolymers of polyethylene oxide-polypropylene oxide- polyethylene oxide (PEO-PPO-PEO). The most preferred coating material is triblock copolymers of polyethylene oxide -polypropylene oxide-

45355640 polyethylene oxide (PEO-PPO-PEO) or poloxamers, also known by the trade name PLURONICs®. Non-limiting examples of poloxamers include PLURONIC® L31, PLURONIC® L35, PLURONIC® L44, PLURONIC® L81, PLURONIC® L101, PLURONIC® L121, PLURONIC ® P65, PLURONIC ® PI03, PLURONIC ® PI05, PLURONIC® PI23,

PLURONIC® L38, PLURONIC® L68, PLURONIC® L87, PLURONIC® L108, PLURONIC® L98, PLURONIC® L127. The polymer referred to by the BASE trade name PLURONIC F127, is called poloxamer 407, and is a hydrophilic non-ionic surfactant of the more general class

of copolymers known as poloxamers. Poloxamer 407 is a trihloek copolymer consisting of a central hydrophobic block of polypropylene glycol flanked by two hydrophilic blocks of polyethylene glycol (PEG). The approximate lengths of die two PEG blocks is 101 repeat units while the approximate length of the propylene glycol block is 56 repeat units. In one embodiment, the particles are coated with polyethylene glycol (PEG).

Poly(ethylene glycol) may be employed to reduce adhesion in brain ECS in certain configurations, e.g., wherein the length of PEG chains extending from the surface is controlled (such that long, unbranched chains that interpenetrate into the ECS are reduced or eliminated). Lor example, linear high MW PEG may be employed in the preparation of particles such that only portions of the linear strands extend from the surface of the particles (e.g., portions equivalent in length to lower MW PEG molecules).

Alternatively, branched high MW PEG may be employed. In such embodiments, although the molecular weight of a PEG molecule may be high, the linear length of any individual strand of the molecule that extends from the surface of a particle would correspond to a linear chain of a lower MW PEG molecule.

The density of the coating can be varied based on a variety of factors including the surface altering material and the composition of the particle. In one embodiment, the density of the surface altering material is at least 0.001, 0.002, 0.005, .008, 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.5, 0.8, 1, 2, 5, 8, 10, 15,

45355640 20, 25, 40, 50, 60, 75, 80, 90, or 100 units per nm ² . The range above is inclusive of all values from 0.001 to 100 units per nm ² .

In another embodiment, the amount of the surface-altering moiety is expressed as a percentage of the mass of the particle. In a particular embodiment, the mass of the surface-altering moiety is at least 1/10,000, 1/7500, 1/5000, 1/4000, 1/3400, 1/2500, 1/2000, 1/1500, 1/1000, 1/750, 1/500, 1/250, 1/200, 1/150, 1/100, 1/75, 1/50, 1/25, 1/20, 1/5, 1/2, or 9/10 of the mass of the particle. The range above is inclusive of all vales from 1/10,000 to 9/10. In another embodiment, the weight percent of the surface altering material is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or greater. The range above is inclusive of all values from 80% to 95%.

4. Particle properties

As shown in the examples, the particles diffuse through the pores of the ECS of the brain at a greater rate of diffusivity than a reference particle, such as an uncoated particle, e.g., uncoated PLGA particle.

The particles described herein may pass through the pores of the ECS of the brain at a rate of diffusivity that is at least 10, 20, 25, 30, 40, 50, 60,

75, 80, 100, 125, 150, 175, 200, 250, 500, 600, 750, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 7500, or 10000- or greater fold higher than a reference particle when measured in vitro. The range above is inclusive of all values from 10 to 10,000.

The transport rates of the particles can be measured using a variety of techniques in the art. In one embodiment, the rate of diffusion is measured by geometric ensemble mean squared displacements (MSD). In a particular embodiment, the particles may diffuse through the pores of the ECS of the brain with an MSD that is at least 5, 20, 30, 50, 60, 75, 80, 100, 125, 150, 200, 250, 500, 600, 750, 1000, 1500, 1750, 2000, 2500, 3000, 4000, 5000, 10000- or greater fold higher than a reference particle. The range above is inclusive of all values from 5 to 10,000.

In other embodiments, the particles diffuse through the pores of the ECS of the brain at a rate approaching the rate of diffusivity at which the

45355640 particles diffuse through water. In a particular embodiment, the rate of diffusivity is at least 1/10,000, 1/7500, 1/5000, 1/1000, 1/800, 1/700, 1/600, 1/500, 1/400, 1/250, 1/200, 1/150, 1/100, 1/75, 1/50, 1/25, 1/10, 1/7, 1/5, 1/2, or 1 times the rate of diffusivity of the particle in water under identical conditions. The range above is inclusive of all values from 1/10,000 to 1.

For example, at a time scale of Is, the rates of diffusion of 40nm, lOOnm, and 200nm uncoated PLGA particles (i.e., unmodified or reference particles) were 76,000-fold, 16,000-fold, and 48,000-fold slower in brain tissue than the same particles in water. In contrast, at a time scale of Is, PLURONIC®- coated particles exhibit 9-fold and 60-fold higher ensemble MSDs in water, respectively, compared with the corresponding uncoated PLGA particles of the same size.

The heterogeneity in particle transport rates can also be evaluated by examining the distribution of individual particle diffusivities at over a particular time period, e.g., Is. Fast moving outlier particles are more likely to penetrate brain tissue and reach greater distances from the point of injection

The particles can be classified based on their mode of transport: diffusive, hindered, or immobile. The x-dependent {MSD} is fitted to the equation {MSD} = 4D ₀ x“, where D ₀ is the t-dependent diffusivity and a is the anomalous diffusion exponent that reflects the extent of impediment. An a = 1 represents unobstructed Brownian diffusion, such as particles in water, where a becomes smaller as obstruction to particle diffusion increases.

Hindered particles have an a value closer to 0, where diffusive particles have an a value closer to 1. In the case where particles experience strong impediment due to interactions with components of the brain ECS, particles typically have an MSD below the microscope detection limit (e.g., lOnm) and are classified as immobile. In one embodiment, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or greater of coated particles of a given average particle size are classified as diffusive. The range of above is inclusive of all values from 15% to 80%.

45355640 The presence of the surface-altering agent can affect the zeta- potential of the particle. In one embodiment, the zeta potential of the particles is -100 mV and 10 mV, between -50 mV and 10 mV, between -25 mV and 10 mV, between -20 mV and 5 mV, between -10 mV and 10 mV, between -10 mV and 5 mV, between -5 mV and 5 mV, or between -2 mV and 2 mV. In a preferred embodiment, the surface charge is neutral or near neutral. The range above is inclusive of all values from -100 mV to 10 mV.

B. Therapeutic, Diagnostic and Prophylactic Agents to be Delivered

The nanoparticles can be used to deliver one or more active agent, particularly one or more active agents to prevent or treat one or more diseases or disorders of the brain. Suitable active agents include therapeutic, diagnostic, and/or prophylactic agents. The agent can be a biomolecule, such as an enzyme, protein, polypeptide, or nucleic acid or a small molecule agent (e.g., molecular weight less than 2000 amu, preferably less than 1500 amu), including organic, inorganic, and organometallic agents. The agent can be encapsulated within the particles, dispersed within the particles, and/or associated with the surface of the particle, either covalently or non- covalently.

Therapeutic agents include chemotherapeutic agents, agents for treatment or alleviation of neurological diseases and disorders, anti inflammatories, agents for treatment of brain trauma, antiinfectives, and combinations thereof.

Exemplary diagnostic materials include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides. Suitable diagnostic agents include, but are not limited to, x-ray imaging agents and contrast media. Radionuclides also can be used as imaging agents.

Examples of other suitable contrast agents include gases or gas emitting compounds, which are radioopaque. Nanoparticles can further include agents useful for determining the location of administered particles. Agents useful for this purpose include fluorescent tags, radionuclides and contrast agents.

45355640 For those embodiments where the one or more therapeutic, prophylactic, and/or diagnostic agents are encapsulated within a polymeric nanoparticle and/or associated with the surface of the nanoparticle, the percent drug loading is from about 1% to about 80%, from about 1% to about 50%, preferably from about 1% to about 40% by weight, more preferably from about 1% to about 20% by weight, most preferably from about 1% to about 10% by weight. The ranges above are inclusive of all values from 1% to 80%. For those embodiments where the agent is associated with the surface of the particle, the percent loading may be higher since the amount of drug is not limited by the methods of encapsulation. In some embodiments, the agent to be delivered may be encapsulated within a nanoparticle and associated with the surface of the particle.

C. Pharmaceutical Excipients for Delivery to the Brain

The particles may be administered in combination with a

physiologically or pharmaceutically acceptable carrier, excipient, or stabilizer. The term“pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term“carrier” refers to an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.

Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. In preferred embodiments, the particles are formulated for parenteral delivery to the brain. Typically the particles will be formulated in sterile saline or buffered solution for injection into the tissues or cells to be treated. The particles can be stored lyophilized in single use vials for rehydration immediately before use. Other means for rehydration and administration are known to those skilled in the art.

45355640 Optional pharmaceutically acceptable excipients include, but are not limited to, lubricants, disintegrants, colorants, stabilizers, and surfactants.

Stabilizers are used to inhibit or retard decomposition reactions which include, by way of example, oxidative reactions.

The nanoparticles or nanoconjugates are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of conjugate appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compositions will be decided by the attending physician within the scope of sound medical judgment. For any nanoparticle or nanoconjugate, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration· Such information can then be used to determine useful doses and routes for administration in humans. Therapeutic efficacy and toxicity of conjugates can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for human use.

III. Pharmaceutical Formulations

Pharmaceutical formulations contain one or more polymeric nanoparticles in combination with one or more pharmaceutically acceptable excipients. Representative excipients include solvents, diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizing agents, and combinations thereof. Suitable pharmaceutically acceptable excipients are preferably selected from materials which are generally recognized as safe

45355640 (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.

Generally, pharmaceutically acceptable salts can be prepared by reaction of the free acid or base forms of an active agent with a

stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Pharmaceutically acceptable salts include salts of an active agent derived from inorganic acids, organic acids, alkali metal salts, and alkaline earth metal salts as well as salts formed by reaction of the drug with a suitable organic ligand (e.g., quaternary ammonium salts). Lists of suitable salts are found, for example, in Remington’s Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704. Examples of ophthalmic drugs sometimes administered in the form of a pharmaceutically acceptable salt include timolol maleate, brimonidine tartrate, and sodium diclofenac.

In some cases, the active agent is a diagnostic agent imaging or otherwise assessing the diseases or conditions of the brain such as brain tumors. Exemplary diagnostic agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, and contrast media.

In certain embodiments, the pharmaceutical composition contains one or more local anesthetics. Representative local anesthetics include tetracaine, lidocaine, amethocaine, proparacaine, lignocaine, and bupivacaine. In some cases, one or more additional agents, such as a hyaluronidase enzyme, is also added to the formulation to accelerate and improves dispersal of the local anesthetic.

A. Formulations for Administration to the Brain

The polymeric nanoparticles will preferably be formulated as a suspension for injection to the brain such as via intracranial administration. In preferred embodiments, the polymeric nanoparticles will preferably be

45355640 formulated suitable for intracranial injection by convection enhanced delivery (CED).

Pharmaceutical formulations for administration to the brain are preferably in the form of a sterile aqueous suspension of nanoparticles. Acceptable solvents include, for example, water, Ringer's solution, phosphate buffered saline (PBS), and isotonic sodium chloride solution. The formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent such as 1,3-butanediol.

In some instances, the formulation is distributed or packaged in a liquid form. Alternatively, formulations for intracranial administration can be packed as a solid, obtained, for example by lyophilization of a suitable liquid formulation. The solid can be reconstituted with an appropriate carrier or diluent prior to administration·

Suspensions or emulsions for intracranial administration may be buffered with an effective amount of buffer necessary to maintain a pH suitable for intracranial administration· Suitable buffers are well known by those skilled in the art and some examples of useful buffers are acetate, borate, carbonate, citrate, and phosphate buffers.

Suspensions, or emulsions for intracranial administration may also contain one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and some examples include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.

Suspensions, or emulsions for intracranial administration may also contain one or more preservatives to prevent bacterial contamination·

Suitable preservatives are known in the art, and include

polyhexamethylenebiguanidine (PHMB), benzalkonium chloride (BAK), stabilized oxychloro complexes (otherwise known as Purite®),

phenylmercuric acetate, chlorobutanol, sorbic acid, chlorhexidine, benzyl alcohol, parabens, thimerosal, and mixtures thereof.

45355640 Suspensions, or emulsions for intracranial administration may also contain one or more excipients known art, such as dispersing agents, wetting agents, and suspending agents.

IV. Methods of Manufacture

A. Particles

Nanoparticles can be made using a variety of techniques in the art. The technique to be used can depend on a variety of factors including the polymer used to form the microparticles and the desired size range of the resulting particles. The type of active agent to be incorporated into the particles may also be a factor as some agents are unstable in the presence of organic solvents and/or high temperatures.

The particles are preferably made using an emulsion technique. Methods for preparing particles include, but are not limited to:

a. Solvent Evaporation. In this method the polymer is dissolved in a volatile organic solvent, such as methylene chloride. The drug (either soluble or dispersed as fine particles) is added to the solution, and the mixture is suspended in an aqueous solution that contains a surface active agent such as poly(vinyl alcohol). The resulting emulsion is stirred until most of the organic solvent evaporated, leaving solid nanoparticles. The resulting nanoparticles are washed with water and dried overnight in a lyophilizer. Nanoparticles with different sizes and morphologies can be obtained by this method. This method is useful for relatively stable polymers like polyesters and polystyrene.

b. Solvent Removal. This technique is primarily designed for poly anhydrides. In this method, the drug is dispersed or dissolved in a solution of the selected polymer in a volatile organic solvent like methylene chloride. This mixture is suspended by stirring in an organic oil (such as silicon oil) to form an emulsion. Unlike solvent evaporation, this method can be used to make nanoparticles from polymers with high melting points and different molecular weights. The external morphology of spheres produced with this technique is highly dependent on the type of polymer used.

45355640 c. Spray-Drying. In this method, the polymer is dissolved in organic solvent. A known amount of the active drug is suspended (insoluble drugs) or co-dissolved (soluble drugs) in the polymer solution. The solution or the dispersion is then spray-dried.

d. Phase Inversion. Microspheres can be formed from polymers using a phase inversion method wherein a polymer is dissolved in a "good" solvent, fine particles of a substance to be incorporated, such as a drug, are mixed or dissolved in the polymer solution, and the mixture is poured into a strong non solvent for the polymer, to spontaneously produce, under favorable conditions, polymeric microspheres, wherein the polymer is either coated with the particles or the particles are dispersed in the polymer. The method can be used to produce nanoparticles in a wide range of sizes, including, for example, about 100 nanometers to about 10 microns.

Exemplary polymers which can be used include polyvinylphenol and polylactic acid. Substances which can be incorporated include, for example, imaging agents such as fluorescent dyes, or biologically active molecules such as proteins or nucleic acids. In the process, the polymer is dissolved in an organic solvent and then contacted with a non solvent, which causes phase inversion of the dissolved polymer to form small spherical particles, with a narrow size distribution optionally incorporating an antigen or other substance.

B. Surface-altering coatings

The particles can be coated with the surface-altering agent using a variety of techniques known in the art depending on whether the coating is covalently or non-covalently associated with the particles. The material can be applied non-covalently, for example, by spray drying.

The coating can be covalently attached to the particles by reacting functional groups on the particles with reactive functional groups on the agent to be attached. For example, am mated PEG can be reacted with reactive functional groups on the particles, such as carboxylic acid groups, to covalently attach the agent via an amide bond. In other embodiments, a

45355640 spacer molecule may be incorporated between the particle surface and the surface- altering agent.

In preferred embodiments, the surface-altering agents are non- covalently associated with the particles.

V. Methods of Use

The particle formulations can be used to administer one or more therapeutic, prophylactic, and/or diagnostic agents directly to the brain to treat one or more diseases or disorders of the brain.

A. Disorders or Diseases to be Treated

In general, these encompass any disease requiring treatments or diagnostic approaches wherein the effect of the active agent(s) would be improved by enhanced tissue penetration, cellular or structural targeting, concomitant delivery, and/or sustained-release, particularly in the brain.

Exemplary diseases and disorders of the brain include, but are not limited to, neoplasms (e.g., cancers, tumors, growths); infections (e.g., HIV/AIDS, Tuberculosis); inflammation (e.g., multiple sclerosis, transverse myelitis and other autoimmune processes, cerebral or tissue edema and other reactive processes); acquired or degenerative conditions (Alzheimer’s disease, Parkinson’s disease, Stroke, Amylotrophic Lateral Sclerosis, Acute and Chronic Traumatic and Pain syndromes); congenital or genetic abnormalities (e.g., Neurofibromatosis, Mucopolysaccaridoses, Tuberous Sclerosis, Von Hippel Lindau); epigenetic conditions, and brain trauma or injury, such as battlefield injuries.

B. Methods of Administration and Dosing

The nanoparticle formulations can be administered using a variety of routes of administration. In some embodiments, the nanoparticle compositions are administered locally to the site/tissue of interest, for example, via direct administration to the brain. Enhanced local delivery can be achieved via convection, electromagnetic, or other forces.

Other modes of administration include intrathecal or intra-ventricular delivery via cerebro- spinal fluid spaces; intra-nasal administration or

45355640 delivery via the olfactory bulb; and systemic delivery via oral, intravenous, or intra-arterial administration.

Enhanced systemic delivery via co- or sequential administration with permeablization agents including but not limited to chemical agents, pharmacologic substances (e.g. cytokines), mechanical barrier disruption (e.g. ultrasound, electron paramagnetic resonance (EPR), ultrasound plus microbubbles), and/or osmotic changes (e.g. mannitol),

In general the timing and frequency of administration will be adjusted to balance the efficacy of a given treatment or diagnostic schedule with the side-effects of the given delivery system. Exemplary dosing frequencies include, but are not limited to, continuous infusion, single and multiple administrations, hourly, daily, weekly, monthly, or yearly dosing.

In preferred embodiments, local delivery into the brain parenchyma is used for enhanced penetration of bioactive or imaging agents in the brain.

Additional factors need to be taken into consideration for optimal delivery of the nanoparticle compositions, for example concentration and osmolality. As described in the examples below, osmolality can affect the distribution. In some embodiments, the osmolality for administration to the brain is between about 50 mOsm/kg, 100 mOsm/kg, 200 mOsm/kg, 300 mOsm/kg, and about 400 mOsm/kg, 500 mOsm/kg, 600 mOsm/kg, 700 mOsm/kg, 800 mOsm/kg, 900 mOsm/kg, orlOOO mOsm/kg; about 200 mOsm/kg to about 600 mOsm/kg, preferably 300 mOsm/kg to 1000 mOsm/kg. The nanoparticles are formulation with an osmolality between about 300 mOsm/kg and about 1000 mOsm/kg, preferably between about 400 mOsm/kg and about 800 mOsm/kg, effective to increase volumetric distribution of the nanoparticles at the site of administration in the brain relative to an equivalent formulation with an osmolality of less than 50 mOsm/kg, or 80 mOsm/kg, or 300 mOsm/kg. In preferred embodiments, hyper-osmolar formulations are used to enhance locally administered nanoparticles in brain parenchyma and/or perivascular space. Synergistic improvement of therapeutic distribution enabled by these non-adhesive, brain-permeable nanoparticles and osmosis-driven brain extracellular matrix

45355640 (ECM) modulation will significantly enhance drug and gene delivery within the CNS, offering higher drug payload, improved drug loading efficiency, and significantly longer drug release durations.

The present invention will be further understood by reference to the following non-limiting examples.

EXAMPLES

Example 1: Physicochemical characterization of PLGA NPs

Surfactants are frequently used to stabilize particle emulsions, enhance drug loading, alter drug release, and change surface properties. As early as 1994, it was demonstrated that nanospheres, synthesized from amphiphilic copolymers composed of two biocompatible blocks (including PEG), exhibit dramatically increased blood circulation times and low liver accumulation in mice. Several PEG-containing block copolymers were then developed and among them, the amphiphilic and polymeric characters of Pluronic® block copolymers such as poloxamer 188 (F68), poloxamer 288 (F98), and poloxamer 407 (F127) have raised growing interest for the design of drug delivery systems. F68 and F127 have been used to coat poly(lactic- co-glycolic acid) (PLGA) NPs to promote blood-brain barrier penetration. They have also been shown to improve efficacy against intracranial gliomas compared to uncoated particles. However, their effect on brain tissue penetration within the brain extracellular matrix (ECM) followed by convection-enhanced delivery (CED) is still unknown, particularly for F98, which has not received the same attention as F68 and F127 in scientific literature.

Pluronic-coated biodegradable NPs present a potential drug delivery platform that could enhance treatments against diffusive brain diseases, particularly, infiltrative glioblastomas, by providing greater brain tissue penetration and broader therapeutic distribution in a safely manner. To this end, the impact of three Pluronic candidates (F68, F98, and F127) was studied in relationship to their physicochemical properties favoring brain penetration followed by CED, using concepts and optimized procedures discussed in previous chapters. PLGA was used since it is one of the most

45355640 successfully developed biodegradable polymers for protection of drug from degradation, sustained release, and possibility to modify surface properties to provide stealthness and/or better interaction with biological materials. The volumetric distribution of the three types of Pluronic-coated PLGA NPs was then compared in relationship to their size and charge, to select the lead NP formulation for further CED optimization and tumor studies.

Methods

PLGA labeling with a fluorescence dye

PLGA polymer (carboxyl terminated, 50:50 monomer ratio)

(Resomer RG 503-H, MW: 30,500) was purchased from Evonik Industries (Darmstadt, Germany) and conjugated with fluorescein cadaverine dye (lEc/lEih: 493/517 nm) (Biotium, Fremont, CA). The reaction was carried out by carbodiimide conjugation, activating carboxyl groups from PLGA for direct reaction with the primary amine via amide bond formation. Briefly, PLGA polymer was dissolved in dimethylformamide (DMF) and activated by N', N-di-cyclohexyl carbodiimide (DCC) and N-hydroxysulfosuccinimide (NHS). The reaction was carried out for 3 hours under constant stirring at room temperature and controlled vacuum with inert N2 gas. Then, pH was adjusted to 7.5 and the fluorescein cadaverine dye was dissolved in DMF and added immediately to the activated polymer solution to react at a polymer:dye molar ratio of 1: 1.2, respectively. This reaction was carried out overnight under constant stirring at room temperature and protected from light. Next, DMF was added to dissolve the precipitates and the solution was filtered by gravity using a Pasteur pipette. Then, the solution was dialyzed for 3 hours against DMF using a Spectra/Por® 6 Standard RC lkD dialysis membrane (Spectrum Laboratories, Inc., Rancho Dominguez, CA). Lastly, DMF was evaporated using a Biotage® V-10 Touch evaporation system (Biotage, Charlotte, NC) and dye conjugation was confirmed by nuclear magnetic resonance (NMR) analysis.

Nanoparticle formulation in ultrapure water

Organic solvents used for preparing polymer solution are known to affect the size of PLGA NPs synthesized through emulsion procedures.

45355640 Partially water-miscible organic solvents— such as benzyl alcohol, butyl lactate, and ethyl acetate— allow nanoparticle formulation through an emulsion-diffusion mechanism and can produce smaller nanoparticles than water-immiscible solvents such as dichloromethane (DCM). Furthermore, Saltzman et al. reported that replacing DCM with ethyl acetate and using the double-emulsion technique improved the yield of small nanoparticles (< 100 nm in diameter).

This protocol was adapted to formulate uncoated PLGA NPs by single-emulsion method. Briefly, fluorescently labeled PLGA polymer was dissolved in acetonitrile (ACN) at 30 mg/ml and 500 pL of the polymer solution was added dropwise into 2 mL of ultrapure water using an SGE syringe (Supelco Inc., Bellefonte, PA). The solution was stirred for 1 hour at room temperature for the primary emulsion, where nanoparticles were formed spontaneously. Then, the emulsion was poured into a beaker with 10 mL of ultrapure water to allow the secondary emulsion to form. This was followed by an additional 4 hours of stirring to allow the solvent to evaporate and particles to harden. After solvent evaporation, the particle solution was washed once with 1 volume of ultrapure water and collected using Amicon Ultra Centrifugal Filters (100,000 MWCO; Millipore Corp., Billerica, MA) at 4,000 x g for 12 minutes until final volume in the filter unit was 500 pL.

Nanoparticle formulation in F68, F98, and FI 27 aqueous solution

Similarly, fluorescently labeled PLGA polymer was dissolved in ACN at 30 mg/ml and 500 pL of the polymer solution was added dropwise into 2 mL of 1% F68, 1% F98, or 1% F127 aqueous solution, using an SGE syringe. The solution was stirred for 1 hour at room temperature for the primary emulsion, where nanoparticles were formed spontaneously. Then, the emulsion was poured into a beaker with 10 mL of 0.5% F68, 0.5% F98, or 0.5% F127 aqueous solution. This was followed by an additional 4 hours of stirring to allow the solvent to evaporate and particles to harden. After solvent evaporation, the particle solution was washed once with 1 volume of ultrapure water and collected using Amicon Ultra Centrifugal Filters

45355640 (100,000 MWCO; Millipore Corp., Billerica, MA) at 4,000 x g for 12 minutes until final volume in the filter unit was 500 pL.

Particle diameter is directly proportional to the polymer concentration (29). Thus, the same protocol was used to formulate F127/PLGA-NPs of various sizes (~60 to 350 nm) by adjusting the PLGA starting concentration to 5, 10, 30, 50, 60, 80 mg/ml in ACN.

Physicochemical characterization of PLGA NPs

The hydrodynamic diameters as well as PDI and z-potentials of uncoated and Pluronic-coated PLGA NPs were measured by DLS and laser Doppler anemometry in 10 mM NaCl solution at pH 7.0, using a Nanosizer ZS90 (Malvern Instruments, Southborough, MA). The size and morphology of nanoparticles were also confirmed by TEM (Hitachi H7600, Japan). Colloidal stability was assessed by monitoring the change in hydrodynamic diameters and PDI in aCSF (Harvard Apparatus, Holliston, MA) at 37 °C.

Statistical Analysis

Statistical analysis between two groups was conducted using a two- tailed Student's t-test assuming unequal variances. If multiple comparisons were involved, one-way analysis of variance (ANOVA), followed by Sidak's multiple comparisons test, was employed, using GraphPad Software (GraphPad Software Inc., La Jolla, CA). Differences were determined to be statistically significant at p < 0.05. Values are presented as mean ± standard error of the mean (SEM).

Results

Pluronic-coated PLGA NPs were formulated by single-emulsion method, mixing the organic PLGA solution into various aqueous Pluronic solutions, F68, F98, and F127. The uncoated PLGA NPs were formed in ultrapure water as a negative control. Hydrodynamic diameter and polydispersity index (PDI) were measured by dynamic light scattering (DLS) in 10 mM NaCl. The z-potential was measured by laser Doppler anemometry in lOmM NaCl at pH 7.0. At least 3 independent experiments were performed for this analysis. FIG.1A shows the hydrodynamic diameters of these PLGA NPs. Table 1 shows that all Pluronic-coated PLGA NPs

45355640 possessed a hydrodynamic diameter of -130 nm and a low PDI -0.1,

whereas uncoated PLGA NPs possessed a slightly larger hydrodynamic diameter of -140 nm and higher PDI of -0.18. On the other hand, the surface charge showed a trend from highly negative to near neutral, as indicated by the z-potential, in the following order: uncoated > F68 > F98 > F127: -50 ± 9 , -26 ± 3 , -9 ± 3 , -4 ± 3 mV , respectively (FIG. IB). The particle morphology was confirmed by transmission electron microscopy (TEM) to be -150 nm spheres regardless of type of coating, consistent with the measured hydrodynamic diameters (Table 1).

Table 1. Physicochemical properties of PLGA NPs with various Pluronic

Z-Average Number mean z-Potential ±

PDI ^a

± SEM (nm) ^a ± SEM (nm) ^a SEM (mV) ^b

Uncoated 180 ± 8 140 ± 6 0.18 -50 ± 9

F68 158 ± 2 126 ± 3 0.09 -26 ± 3

F98 162 ± 5 128 ± 4 0.12 -9 ± 3

F127 161 ± 1 134 ± 3 0.07 -4 ± 3 ^a Hydrodynamic diameter and polydispersity index (PDI) were measured by dynamic light scattering (DLS) in 10 mM NaCI at pH 7.0. Mean ± SEM (n > 3).

^-potential was measured by laser Doppler anemometry in 10 mM NaCI at pH 7.0. Mean ± SEM (n > 3).

The hydrodynamic diameters of the various PLGA NPs were

assessed following a 12-hour incubation in artificial cerebrospinal fluid

(aCSF) at 37°C to predict potential alteration of particle properties in the physiological brain environment. All formulations retained their

hydrodynamic diameters and PDI in aCSF over time. Notably, F98 showed less colloidal stability than the other groups, but even with the drastic and sudden changes, it retained the diameter in similar range as the other PLGA NPs (FIG.1C).

45355640 Example 2: Volume of distribution in healthy rat brains

Methods

Animal studies

6-to-8-week-old female Fischer 344 rats were used for the assessment of the volume of distribution of the various PLGA NPs. Animals were treated in accordance with the guidelines and policies of the Johns Hopkins University Animal Care and Use Committee. Surgical procedures were performed using standard sterile surgical techniques. Animals were anesthetized using a mixture of 75 mg/kg ketamine and 7.5 mg/kg xylazine. A midline scalp incision was made to expose the coronal and sagittal sutures and a burr whole was drilled 3 mm lateral to the sagittal suture and 0.5 mm posterior to the bregma. Following the tumor cell inoculation or PLGA NPs administration, the skin was sealed using biodegradable sutures (Polysorb™ Braided Absorbable Sutures 5-0) and bacitracin was applied.

Intracranial administration - CED

To assess the volume of distribution of the various PLGA NPs, fluorescently labeled NPs were intracranially administered via CED. Briefly, a Neuros syringe connected to a 33-gauge needle was filled with 20 pL of PLGA NPs solution at a polymer concentration of 15 mg/mL and lowered to a depth of 3.5 or 2.5 mm of a healthy or an orthotopic tumor-bearing rat brain, targeting the striatum or the tumor core, respectively. PLGA NPs were then infused at a rate of 0.5 pL/min as controlled by a Chemyx Nanojet Injector Module (Chemyx, Stafford, TX).

Imaging and quantification of volumetric of distribution

Brains were harvested immediately after PLGA NPs administration and flash-frozen in dry-ice. Tissues were then sectioned using a Leica CM 1905 cryostat into 100 or 50 pm coronal slices for healthy or tumor-bearing rats at ± 3 mm of the infusion plane in striatum or until the tumor tissue was no longer visible, respectively. All fluorescence images were taken using an LSM 710 confocal microscope under 5X magnification. The settings were optimized to avoid background fluorescence based on the microscopy of untreated control rat brains. The volume of distribution was quantified using

45355640 a custom-made MATLAB script that subtracted background fluorescence by Otsu’s method of thresholding. The area of PLGA NPs coverage in each slice was integrated to calculate the total volume of distribution.

To reconstruct 3D-rendered imaginings of F127/PLGA-NPs distribution in orthotopic tumor, we stacked and aligned the acquired images using Metamorph ^® Microscopy Automation & Image Analysis Software (Molecular Devices, CA). Imaris Software (Bitplane, CT) was used to create 3D isosurfaces.

Results

The various PLGA NPs were administered into the rats’ striatum via CED, which has been clinically applied to enhance the therapeutic distribution within brain by creating a continuous pressure-driven bulk flow. The volumetric distribution at the infusion site of PLGA NPs when uncoated and coated with F68, F98, F127 in healthy rat brains obtained by confocal microscopy immediately after the treatment was then assessed. Images showing the coronal plane at the site of administration revealed enhanced volumes of distribution for all Pluronic-coated PLGA NPs, whereas the uncoated control was confined around the needle track. The trend seen in the volume of distribution of PLGA NPs in rat brains was inversely correlated to their surface charge: F127 > F98 > F68 > uncoated. Quantitatively, F127/PLGA-NPs exhibited significantly greater volumetric distribution compared to all the groups (FIG. 2A), with an average volumetric distribution of 38 ± 5 mm ³ , 23 ± 3 mm ³ , 13 ± 0.7 mm ³ , 4 ± 0.7 mm ³ , for F127, F98 (p <0.05), F68 (p < 0.001), uncoated (p < 0.001), respectively. Notably, F98/PLGA-NPs showed the second highest value for volume of distribution, exhibiting statistical significance when compared to F68 (p < 0.05) and uncoated (p < 0.01) NPs.

It was also confirmed that the two best Pluronic candidates, F98 and FI 27, were still present in the brain parenchyma 48 hours after NPs treatment, albeit, the volume of distribution decreased by ~2-fold when compared to the volume of distribution achieved immediately after CED. Quantitatively, F98 showed a volumetric distribution of 10 ± 2 mm ³ ,

45355640 whereas FI 27 demonstrated significantly greater distribution than F98 by ~2- fold, with 22 ± 3 mm ³ (p < 0.05) (FIG.2B).

Example 3: Effect of polymer concentration on F127/PLGA-NPs

Results

Next, the effect of PLGA concentration on the physicochemical properties of the F127/PLGA-NPs was evaluated. The data of the various types of PLGA NPs was taken from batches at a starting polymer concentration of 30 mg/ml. This same protocol was then adjusted to formulate F127/PLGA-NPs of various sizes (~60 to 350 nm) by changing the PLGA starting concentration to 5, 10, 30, 50, 60, 80 mg/ml. Here, the batch of F127/PLGA-NPs at a starting concentration of 30 mg/ml was used as a control. The highest polymer concentration that provided consistent NPs yield, according to DLS, was 80 mg/ml; concentrations greater than that one did not allow successful recovery during the washing process due to significant polymer aggregation. See Fig. 3.

Table 2 shows a tendency consistent with the expected results, where increments of the starting PLGA concentration yielded larger diameters. Although the PDI was not significantly affected by the polymer

concentration, the surface charge showed a“more negative” inclination as the PLGA concentration increased. Note, we named each condition based on the hydrodynamic diameter after the number mean reported by DLS. For example,“F127/PLGA-130” refers to the control batch of 30 mg/ml, which showed a hydrodynamic diameter of 135 ± 5 nm. Lastly, the colloidal stability of the various F127/PLGA-NPs was assessed (only four are shown in FIG.3) and confirmed that the hydrodynamic diameters did not change over time in physiological conditions.

45355640 Table 2. Effect of polymer concentration on physiochemical properties of

Polymer

-Average Number mean z-Potential

F127/PLGA-NPs concentratio

SEM (nm) ^a ± SEM (nm) ^{a PDI} ± SEM (mV) ^b

(mg/ml)

5 89 ± 2 63 ± 3 0.07 ± 0.05 1 .6 6.2

1 0 111 ± 8 89 ± 8 0.08 ± 0.04 -2.2 ± 5.8

30 160 ± 7 135 ± 5 0.08 ± 0.03 -3.5 ± 4.5

50 198 ± 9 171 ± 13 0.10 ± 0.02 -4.0 ± 4.2

60 257 ± 1 198 ± 4 0.16 ± 0.02 -4.6 ± 4.3 80 387 ± 14 341 ± 36 0.10 ± 0.04 -4.8 ± 4.1

Hydrodynamic diameter and PDI were measured by DLS in 10 mM NaCI ± SEM (n > 3).

b

z-potential was measured by laser Doppler anemometry in 10 mM NaCI at pH 7.0.

Mean ± SEM (n > 3).

Example 4: Effect of changes in diameter on in vivo volume of

distribution

Results

Cationic polymer-based NPs have a“sticky” limitation when it

comes to brain penetration, which is due to the adhesive moieties of brain

ECM and other brain-related barriers. Also, NPs larger than -120 nm cannot

efficiently diffuse in ex vivo and in vivo brain tissue even after PEGylation.

To this end, it was evaluated whether F127/PLGA-NPs possessing large

diameters and a near neutral, yet slightly negative, surface charge can still

achieve widespread distribution when combined with the CED technique.

For this study, four of the six groups were selected from Table 2,

F127/PLGA- 60, 130, 170, and 200 nm. Figure 4 showed that the

widespread distribution is inversely correlated with the hydrodynamic

diameter. Quantitatively, there was no significant difference between the

particles in the smaller range, 60 nm and 130 nm, showing 40 ± 4 mm ³ and

38 ± 5 mm ³ , for the volume of distribution, respectively. Similarly, the

particles in the larger range, 170 nm and 200 nm, achieved comparable

distribution, showing 25 ± 5 mm ³ and 24 ± 3 mm ³ , respectively. Both, 60

and 130 nm NPs demonstrated statistical significance when compared to 200

nm NPs (p < 0.05) but not against 170 nm NPs.

45355640 Example 5: Histopathological analysis and safety profile

Methods

The toxicity of F98/PLGA NPs and F127/PLGA NPs was compared to medical-grade normal saline (NS) by histology safety profile in the rats’ striatum following intracranial administration via CED of Pluronic-coated PLGA NPs,. Animals were sacrificed 3 days following administration and the harvested brains were fixed in 4% formaldehyde, processed, sectioned and stained with hematoxylin and eosin (H&E) by the Johns Hopkins Reference Histology Laboratory. The point of infusion was identified by the tissue cavity imparted by the needle and the region immediately adjacent was imaged and evaluated. Blind histopathological analysis was performed by a board-certified neuropathologist and tissues were scored from 0-3 for indications of inflammation and hemorrhage (0: no

inflammation/hemorrhage, 1: mild, 2: moderate, 3: severe).

Results

To demonstrate the safety of Pluronic-coated PLGA NPs followed by intracranial administration via CED, the toxicity of F98/PLGA NPs and F127/PLGA NPs was compared to medical-grade normal saline (NS) by histology safety profile in the rats’ striatum. Following 3 days from the NPs administration, the average toxicity scores of the brain tissues for both Pluronic coatings were comparable to those of NS-treated controls (F1G.5), with an average score of -1.5-2 for hemorrhage and inflammation, showing that they were well-tolerated at the dose administered (15 mg/mL, 20 pL). Notably, the inflammation score increased with the respective volume of distribution in the brain (F127 > F98). Importantly, regardless of the type of particle, inflammatory and hemorrhagic changes were confined around the injection site and did not propagate throughout the brain tissue. All hemorrhage and inflammation scores were negligible, -0-1, after 30 days.

45355640 Example 6: Widespread distribution in orthotopic brain tumors

Methods

Orthotopic tumor inoculations

Orthotopic tumor cell inoculations were. Briefly, 100,000 cells of rat glioblastoma cells expressing mKate fluorescent protein (F98-mKate), were administered in 10 pL of DMEM over 5 minutes at a depth of 3.5 mm using a Neuros Syringe (50 pL; Hamilton, Reno, NV) mounted on an ultra-precise small-animal stereotactic frame. Rat brains were inoculated with a high numbers of tumor cells, relevant to clinical translation, to establish a model mimicking malignant gliomas characterized by rapid tumor growth rates.

Results

It was evaluated whether F127/PLGA-NPs can effectively diffuse and penetrate the dense cellularity within an orthotopic model of aggressive brain tumor following CED. The model was established by intracranial stereotactic inoculation of 1 x 10 ⁵ F98 cells and a CED experiment was conducted 10 days after the inoculation when a large volume (~50 mm ³ ) of orthotopic tumor was established in the rat striatum. As shown by representative 3D-reconstructed images (FIG.6A and FIG.6B), F127/PLGA- NPs exhibited almost 100% tumor coverage, with widespread distribution inside and outside the tumor core (FIG.6B). Highly invasive tumor cells that migrate beyond the tumor edge and infiltrate into normal brain tissue are responsible for recurrence of malignant gliomas. Thus, the unique ability of F127/PLGA-NPs to efficiently penetrate both normal brain parenchyma and brain tumor tissue may serve favorably for this specific application.

Example 7: Lyophilization and cryoprotection of F127/PLGA-NPs

Methods

Lyophilization and administration of the“ready-to-use” F127/PLGA

NPs

For lyophilized F127/PLGA-NPs formation and evaluation, NPs were initially formed as described above at two different PLGA concentrations, 5 and 30 mg/ml. Subsequently, sucrose (OPS Diagnostics, Lebanon, NJ) was added as a cryoprotectant to a final concentration of 30 mg/mL, diluting the

45355640 solution of F127/PLGA-NPs by 2-fold. Particles were then frozen in dry-ice and lyophilized overnight. Lyophilized F127/PLGA-NPs were stored at -20 °C until use and then reconstituted in sterile ultrapure water and used at the same concentration as freshly prepared F127/PLGA-NPs.

To assess the effect of osmolality of infusate solutions on the distribution of the“ready-to-use” F127/PLGA-NPs in the brain parenchyma, the osmotic pressures were adjusted by addition of NaCl to a final osmolality of 300 mOsm/kg, 370 mOsm/kg, and 480 mOsm/kg and intracranially administered via CED. The CED and imaging procedure were executed as described above.

Results

Pluronic-coated PLGA NPs are stable at room temperature in aqueous solution. They can retain their colloidal stability for weeks and even months. However, without proper care, the polymer concentration can be affected over time due to water evaporation, which could lead to“gel like” consistency. Therefore, lyophilized NPs are more convenient for handling, as they the user simply needs to add water to adjust the desired concentration and inject. To ensure that F127/PLGA-NPs do not lose their nanoparticle characteristics after lyophilization, the physicochemical properties of two different types of F127-coated NPs, 60 nm and 130 nm, with and without sucrose as a cryoprotectant, were compared.

As Table 3 shows, particles without sucrose do not retain the original size and charge, whereas particles properly mixed with sucrose kept comparable values as the original solution.

45355640 Table 3. Physiochemical properties of F127/PLGA NPs after

Z-Average Number mean _pn|a z-Potential +

F127/PLGA NPs Cryoprotectant

+ SEM (nm) ^a + SEM (nm) ^a SEM (mV) ^b

F127/PLGA-60 N/A 124 ± 3 93 ± 2 0.14 ± 0.03 -3.7 ± 8.8

F127/PLGA-60 ucrose 93 ± 5 52 ± 4 0.17 ± 0.04 -1 .9 ± 6.9

F127/PLGA-130 N/A 549 ± 135 180 ± 97 0.49 ± 0.12 -4.5 ± 3.5

F127/PLGA-130 ucrose 170 ± 6 127 ± 2 0.10 ± 0.02 -5.3 ± 5.1

Example 8: Effect of osmolality on in vivo volume of distribution

Materials and Methods

Administration of hyperosmolar saline in brain tissue results in enlargement of the ECM mesh spacings as water is drawn out of cells into intercellular space via an osmotic gradient established by the hyperosmolar saline.To verify that the osmotic modulation of the intercellular space translates in vivo, lyophilized F127/PLGA-NPs was administered via CED in saline-based infusate solutions with varying osmolality and determined their effects on the volume of distribution. Fresh particles were infused at 300 mOsm/kg (iso-osmolar, 0.9 % saline) as a control, yielding 41 ± 4 mm ³ . Results

The volume of distribution was positively correlated with the osmolality of the infusate solution (FIG.7). Quantitively, NPs infused at 480 mOsm/kg (hyper-osmolar) exhibited significantly greater volume of distribution, showing 69 ± 6 mm ³ , compared to 370 and 300 mOsm/kg, which exhibited 63 ± 3 mm ³ and 45 ± 5 mm ³ , respectively.

Pluronic-coating enhances NPs physicochemical properties for brain penetration, including colloidal stability and near-neutral surface charge. The size and charge can be manipulated as necessary by adjusting the starting PLGA concentration but keeping into consideration that the volumetric distribution is inversely correlated to the hydrodynamic diameter, without altering the colloidal stability. This demonstrates that the“mesh-like” pores sizes of the brain tolerate a dynamic equilibrium when slow pressure gradients occur, which allow“expansion” and“flexibility” for enough penetration when larger, but well coated, NPs are infused via CED.

45355640 F127/PLGA-NPs provided the highest volume of distribution in healthy brain parenchyma, with great particle retention after 48 hours from CED. F127/PLGA-NPs can efficaciously achieve widespread distribution into a highly aggressive orthotopic brain tumor model when combined with CED. Altering the pore sizes of the rat brain ECM by using lyophilized NPs with infusate solutions of varying osmolality, improved the volumetric distribution in vivo. The combination approach of the“ready-to-use” F127/PLGA-NPs, administered in hyper-osmolar infusate solutions via CED, may be widely employed to promote widespread delivery of therapeutics throughout the brain.

The CED approach overcomes a major obstacle hindering the development of drug therapies, i.e. poor local diffusion, by allowing slower infusion rates and controlled pressure gradients in the brain parenchyma. However, the CED technique is not enough for achieving widespread distribution. Poloxamers such as F127, F98, and F68, consist of a hydrophobic polypropylene oxide (PPO) block and hydrophilic polyethylene oxide (PEO) segments, where the composition (PPO:PEO) and MW (block length) impact the properties of the surfactant. Without Pluronic coatings, PLGA NPs have exposed hydrophobic regions on the particle core and z- potentials more negative than -40 mV. These particles adhered to

components within the brain parenchyma by electrostatic or hydrophobic interactions, limiting the diffusive nature of the particles even after CED. Although the significant differences in the surface charge of the various types of PLGA NPs do not affect the colloidal stability under physiological conditions, widespread distribution remains inversely correlated with the z- potentials. Since F127 has a longer PPO segment compared to F98 and F68, it allowed better shielding of any exposed hydrophobic regions.

Consequently, FI 27 demonstrated better volumetric distribution in healthy brain tissues than F98, F68, and uncoated PLGA NPs, respectively.

The pore sizes of the brain ECM has been previously estimated to be -38 - 64 nm, but it has been recently re-evaluated them using non-adhesive probes to be as large as -110 nm. A lot of studies have combined the CED

45355640 approach with nanocarriers possessing -100 - 150 nm in size to treat brain tumors. Very few studies have used the CED technique to infuse

nanocarriers with larger diameters, probably due to the known size-constraint in the ECM. As an example, MacKay J. A. et al. reported that -200 nm liposomes penetrated less than 80 nm liposomes even when shielded with PEG. Similarly, Zhan W. et al. reported that liposomes with 100 nm and 135 nm in diameter have comparable diffusivity in tumor tissue, whereas the value reduces by one order of magnitude when diameter reaches 500 nm.

This is consistent with what was observed when infusing F127/PLGA-NPs with diameters larger than -160 nm, which experienced a reduction in the volumetric distribution by ~2-fold after CED. Based on these results, it was determined that non-adhesive NPs, smaller than -150 nm, are preferable for maximum brain tissue penetration, particularly for brain tumor applications.

The unique pathological hallmarks of brain tumors, particularly malignant gliomas, present significant challenges for CED. First, brain tumors are often characterized by the presence of necrotic regions, hemorrhage and fibrin clots, making them naturally heterogeneous structures. Second, the increased vascular permeability in tumors and surrounding regions can cause rapid clearance of infused drugs.

Additionally, the high pressure within brain tumors can lead to reflux or backflow of the infusate up through the catheter. These features have shown to affect distribution of polymer-based NPs after CED. Therefore, nanoparticle distribution depends on the size and properties of the tumor and appears to be heterogeneous and asymmetric. Saucier-Sawyer J. K. et al. showed how significant quantities of PLGA NPs tend to accumulate in the peritumoral space, such that most of the tumor is surrounded by particles which are distributed along the tumor margins. In these studies, images from F127/PLGA-NPs infused into large F98 tumors showed consistent coverage of the tumor, where the margins of these larger tumors are surrounded by the “excess” of particles that diffused further away from the tumor core due to the“human-error” of the CED execution. These findings suggest that tumor

45355640 size and NPs characteristics are important criteria that should be considered when determining infusion site and parameters for CED.

The steric obstruction imposed by the ECM structure remains a challenging limitation to non-adhesive NPs for achieving widespread distribution in the brain, particularly when relatively large NPs are required. To overcome this hurdle, Nicholson et al. and Zhang C. et al. have demonstrated that exposure of brain tissues to modestly hyperosmolar solutions (500 mOsmol/kg) increases the volume of the intercellular space, which minimizes the tissue's resistance to NP diffusion. In agreement with these studies, it was demonstrated that the osmotic modulation of the“ready- to-use” F127/PLGA-NPs resulted in enhanced volume of distribution, by enlargement of the ECM mesh spacings as water is drawn out of cells into intercellular space via an osmotic gradient established by the hyperosmolar saline.

In this study, it has been demonstrated that the use of Pluronic coatings improves the ability of the most- widely used biodegradable PLGA nanoparticle platform to penetrate within the brain parenchyma. This led to the discovery of a new brain-penetrating nanoparticle formulation,

F127/PLGA-NPs, for future drug studies in brain tumors. A simple and safe method to maximize the distribution of therapeutic F127/PLGA-NPs in the brain combines the CED approach in a hyperosmolar infusate solution, that could address major drawbacks currently associated with CED applications in clinical trials, including limited ECM distribution and perivascular sequestration.

Prior studies have demonstrated that administration of hyperosmolar saline in brain tissue results in enlargement of the ECM mesh spacings as water is drawn out of cells into intercellular space via an osmotic gradient established by the hyperosmolar saline. To verify that the osmotic modulation of the intercellular space translates in vivo, lyophilized

F127/PLGA-NPs via CED in saline-based infusate solutions with varying osmolality and determined their effects on the volume of distribution. Fresh particles were infused at 300 mOsm/kg (iso-osmolar, 0.9 % saline) as a

45355640 control, yielding 41 ± 4 mm ³ . The volume of distribution was positively correlated with the osmolality of the infusate solution (Figure 7).

Quantitively, NPs infused at 480 mOsm/kg (hyper-osmolar) exhibited significantly greater volume of distribution, showing 69 ± 6 mm ³ , compared to 370 and 300 mOsm/kg, which exhibited 63 ± 3 mm ³ and 45 ± 5 mm ³ , respectively.

Summary of Results and Conclusions

Pluronic-coating enhances NPs physicochemical properties for brain penetration, including colloidal stability and near-neutral surface charge. The size and charge can be manipulated as necessary by adjusting the starting PLGA concentration but keeping into consideration that the volumetric distribution is inversely correlated to the hydrodynamic diameter, without altering the colloidal stability. This demonstrates that the“mesh-like” pores sizes of the brain tolerate for a dynamic equilibrium when slow pressure gradients occur, which allow“expansion” and“flexibility” for enough penetration when larger, but well coated, NPs are infused via CED.

F127/PLGA-NPs provided the highest volume of distribution in healthy brain parenchyma, with great particle retention after 48 hours from CED. F127/PLGA-NPs can efficaciously achieve widespread distribution into a highly aggressive orthotopic brain tumor model when combined with CED. Alteration of the pore sizes of the rat brain ECM by using lyophilized NPs with infusate solutions of varying osmolality, improved the volumetric distribution in vivo. The combination approach of the“ready-to-use” F127/PLGA-NPs, administered in hyper-osmolar infusate solutions via CED, may be widely employed to promote widespread delivery of therapeutics throughout the brain.

The CED approach overcomes a major obstacle hindering the development of drug therapies, i.e. poor local diffusion, by allowing slower infusion rates and controlled pressure gradients in the brain parenchyma. Alone, the CED technique is not enough for achieving widespread distribution. Poloxamers such as F127, F98, and F68, consist of a hydrophobic polypropylene oxide (PPO) block and hydrophilic polyethylene

45355640 oxide (PEO) segments, where the composition (PPO:PEO) and MW (block length) impact the properties of the surfactant. Without Pluronic coatings, PLGA NPs have exposed hydrophobic regions on the particle core and z- potentials more negative than -40 mV. These particles adhere to components within the brain parenchyma by electrostatic or hydrophobic interactions, limiting the diffusive nature of the particles even after CED. Although the significant differences in the surface charge of the various types of PLGA NPs do not affect the colloidal stability under physiological conditions, widespread distribution remains inversely correlated with the z-potentials. Since F127 has a longer PPO segment compared to F98 and F68, it allowed better shielding of any exposed hydrophobic regions. Consequently, F127 demonstrated better volumetric distribution in healthy brain tissues than F98, F68, and uncoated PLGA NPs, respectively.

The pore sizes of the brain ECM has been previously estimated to be -38 - 64 nm, but have now been shown using non-adhesive probes to be as large as -110 nm. Infusing F127/PLGA-NPs with diameters larger than -160 nm showed they experienced a reduction in the volumetric distribution by ~2-fold after CED. Based on these results, it was determined that non adhesive NPs, smaller than -150 nm, are preferable for maximum brain tissue penetration, particularly for brain tumor applications.

The unique pathological hallmarks of brain tumors, particularly malignant gliomas, present significant challenges for CED. First, brain tumors are often characterized by the presence of necrotic regions, hemorrhage and fibrin clots, making them naturally heterogeneous structures. Second, the increased vascular permeability in tumors and surrounding regions can cause rapid clearance of infused drugs.

Additionally, the high pressure within brain tumors can lead to reflux or backflow of the infusate up through the catheter. These features have shown to affect distribution of polymer-based NPs after CED. Therefore, nanoparticle distribution depends on the size and properties of the tumor and appears to be heterogeneous and asymmetric. Significant quantities of PLGA NPs tend to accumulate in the peritumoral space, such that most of the tumor

45355640 is surrounded by particles which are distributed along the tumor margins. In contrast, images from F127/PLGA-NPs infused into large F98 tumors showed consistent coverage of the tumor, where the margins of these larger tumors are surrounded by the“excess” of particles that diffused further away from the tumor core due to the“human-error” of the CED execution. These findings show that tumor size and NPs characteristics are important criteria that should be considered when determining infusion site and parameters for CED.

45355640