Patent Application
20200016088
The present disclosure relates to compositions and methods to treat neurological disorders. In exemplary embodiments, compositions are disclosed that are designed to cross the blood-brain barrier and control the release of a compound.
The development of new treatments for diseases of the central nervous system (CNS) is a pressing public health concern [1-4] (references in [brackets] refer to reference citations set forth hereinbelow). To treat neurological diseases in the brain, one route of treatment is delivering beneficial therapies, such as neuropeptides, across the blood-brain barrier (BBB) and into the brain. Neuropeptides are small protein-like molecules that are commonly co-released with neurotransmitters to modulate neurotransmission. Neuropeptides vary in their size, the neuroanatomical distribution of the neurons in which they are synthesized, and in the neurotransmitters with which they partner for co-release. They have shown particular promise as potential therapeutics for CNS disorders as observed in animal studies that demonstrate profound efficacy in treating a number of disease states. Although there are many neuropeptides with potential therapeutic benefits, one of particular interest is oxytocin (OT). OT is a nine-amino acid neuropeptide that is synthesized in the paraventricular and supraoptic nuclei of the hypothalamus. It is released into peripheral circulation by the posterior pituitary gland and also functions as a neurotransmitter throughout much of the mammalian brain [5]. Although OT has been traditionally recognized to be involved in uterine stimulation during parturition, recent studies have convincingly demonstrated its roles for the brain neurotransmitter OT system in social behavior, social recognition, social comfort, and altruism [6-8].
Unfortunately, a fundamental obstacle for the development of OT as a CNS medication is that it (1) does not readily passively cross the BBB following exogenous administration and (2) its half-life in the blood is short, on the order of minutes. The BBB limits large or hydrophilic molecules from readily entering the brain. This limits the range of compounds and scaffolds available to medicinal chemistry programs in the development of new chemical entities for treating CNS disorders. One method to overcome the resistance of the BBB is surgical cannulation of the brain ventricles for widespread or region-specific brain delivery of large molecule compounds to treat CNS disorders in animal models. Some of these compounds include neurotrophins such as brain-derived neurotropic factor (BDNF) [9-10], secretase inhibitors for Alzheimer's disease [11], anti-inflammatory cytokines [12-13], and therapeutic plasmid DNA for neuronal stimulation or myelin recovery and remodeling [14-16]. However, delivering these molecules by surgical cannulation is an invasive procedure. Alternatively, researchers have tried to utilize the mechanisms present in the BBB to deliver compounds to the brain without physical or surgical disruption of the barrier. Poor target penetrance and short half-lives are key factors that limit the ability to administer neuropeptides as therapies. A viable approach for large-molecule delivery to the CNS that does not require direct drug application would advance these compounds towards the clinic.
Receptor-mediated transport (RMT) [17] is a class of transport system that transports macromolecules such as insulin and transferrin between the blood and the brain. Transferrin (Tf) has been widely studied and shown to be a promising molecular probe for targeted drug delivery to the brain through RMT as transferrin receptors are known to be expressed in the luminal membrane of capillary endothelium of the BBB [18-20]. Tf is a single chain 80 kDa protein that facilitates the movement of iron between the blood and brain. Tf-bound iron from blood binds to the Tf receptor (TfR) expressed on the BBB. The TfR is expressed mostly on endothelial cells, intestinal cells, hepatocytes, and monocytes. Tf-conjugated drug delivery systems (nanoparticles, liposomes and micelles) can improve drug transport across the BBB since Tf-receptors (TfR) are overexpressed in brain capillary endothelium [21-26]. Previous studies have extensively examined the Transferrin (Tf) system as a mean of targeting drug formulations to the brain [22, 23, 27, 28]. Tf can be adsorbed on or conjugated to different polymers including PLGA, polyethylene glycol, lipopolyplexes, polymer-based cyclodextrin, and gold nanoparticles [29-32]. Recent studies have also described a new brain targeting ligand called Rabies Virus Glycoprotein (RVG), which putatively binds to nicotinic cholinergic receptors on the BBB to engage brain uptake of drug formulations [33]. Both Tf and RVG have been used previously to target therapeutic peptides to the brain, and are as such likely to be effective in the transport of neuropeptides into the brain.
The following presents a simplified summary in order to provide a basic understanding of some aspects of various invention embodiments. The summary is not an extensive overview of the invention. It is neither intended to identify key or critical elements of the invention nor to delineate the scope of the invention. The following summary merely presents some concepts of the invention in a simplified form as a prelude to the more detailed description below.
Generally described, the present disclosure provides in a first exemplary embodiment a composition comprising an active material; a nanoparticle, wherein the nanoparticle encapsulates the active material; and, at least one targeting ligand, wherein the at least one targeting ligand is coupled to the nanoparticle.
In another embodiment, the composition comprises an active material, wherein the active is a neuropeptide.
In another embodiment the composition comprises an active material, wherein the active material is oxytocin.
In another embodiment the composition of comprises a nanoparticle, wherein the nanoparticle is comprised of BSA.
In another embodiment the composition comprises at least one targeting ligand, wherein the at least one targeting ligand is RVG. In another embodiment the RVG is adsorbed onto the nanoparticle.
The disclosure also generally includes a method of treating a neurological disorder comprising administering a composition described herein to a patient. In another embodiment the method includes treating a neurological disorder that is a central nervous system disorder. In another embodiment the neurological disorder is Autism Spectrum disorder.
In another embodiment the composition to treat a neurological disorder is administered intranasally.
Other features will become apparent upon reading the following detailed description of certain exemplary embodiments, when taken in conjunction with the appended claims.
The drawings disclose exemplary embodiments in which like reference characters designate the same or similar parts throughout the figures of which:
Compositions and methods are disclosed for targeting, penetrating, or crossing the blood-brain barrier (BBB). Further, compositions and methods are disclosed for treating a neurological disorder in a patient.
In one exemplary embodiment, a composition is administered to a patient to treat a neurological disorder. In accordance with the disclosure, “patient” may refer to a human or an animal. Accordingly, the methods and compositions disclosed herein can be used, or adapted for use with both human clinical medicine and veterinary application.
In one exemplary embodiment, an active material is encapsulated in at least one nanoparticle, wherein the nanoparticle is coated in targeting ligands.
In one exemplary embodiment, nanoparticles can be used to encapsulate an active material. Further, the active material can be a target neuropeptide and the nanoparticles encapsulating the target neuropeptide can be coated with a targeting ligand (e.g., Tf or RVG) to utilize receptor-mediated transport. One type of receptor-mediated transport is referred to as transcytosis and can be used to move an active material across the BBB. Receptor mediated transcytosis is a class of transport system that transports macromolecules like insulin and Tf between blood and brain. Nanoparticles having sizes smaller than 500 nm are usually taken via the clathrin-mediated transport system whereas larger particles are usually transported via micropinocytosis [54]. In reference to charge, negatively-charged nanoparticles are endocytosed by interacting with the positive site of the proteins on a membrane.
In another exemplary embodiment, the active material can be encapsulated within different polymers including poly(lactic-co-glycolic acid) (PLGA), Bovine serum albumin (BSA), polyethylene glycol, lipopolyplexes, polymer-based cyclodextrin, and gold nanoparticles [29-32]. Additionally, smaller nanoparticles (e.g., under 200 nm in diameter) can potentially increase the penetrance of the formulation through the BBB and into the brain. In further exemplary embodiments, the nanoparticles can range in an average size of up to 200 nm in diameter. In other exemplary embodiments, the nanoparticles can range in an average size of less than 200 nm in diameter. In other exemplary embodiments the nanoparticles' diameters can range in an average size of about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 155 nm, about 160 nm, about 165 nm, about 170 nm, about 175 nm, about 180 nm, about 185 nm, about 190 nm, about 195 nm, about 200 nm, about 205 nm, and about 210 nm. Furthermore, both PLGA and BSA nanoparticles have sustained release properties, which can increase the half-life of an active material (e.g., oxytocin (OT)) and thereby decrease the frequency of administration, which can increase patient compliance. In other exemplary embodiments, a composition comprising PLGA or BSA nanoparticles have a release profile that provides an initial burst release of an active material (e.g., a peptide) to induce rapid symptom control followed by a sustained release for prolonged therapeutic effects.
In one exemplary embodiment the active material include can one or more of the following: small molecules, biologics, peptides, neuropeptides, macromolecules, antibodies or fragments thereof, ligands, DNA, RNA, proteins, fluorescent markers, or a combination or mixture of at least two of the foregoing. In certain exemplary embodiments, a neuropeptide is disclosed. In further exemplary embodiments, the neuropeptide is oxytocin (OT).
In another exemplary embodiment, the composition is used to treat a neural disorder. In some embodiments the neural disorder can be a central nervous system (CNS) disorder with social deficit components, including Autism Spectrum Disorder (ASD) and Dravet syndrome.
In one exemplary embodiment, two polymeric-based nanoparticle formulations were developed in order to increase the brain penetrance and extend the duration of action of OT.
For the first formulation, the OT-loaded PLGA particles were prepared by dissolving poly(lactic-co-glycolic acid) (PLGA) in dichloromethane, adding the OT along with sorbitan monooleate, homogenizing the particles in a water/oil emulsion, and adding poly vinyl alcohol to create a final water/oil/water (w/o/w) emulsion.
The second formulation, OT-loaded BSA-crosslinked particles were created by a coacervation/nanoprecipitation technique (see further details in the Examples hereinbelow; all references to “Examples” refer to the Examples described hereinbelow) to encapsulate OT and form the particles.
Next, the physical, physiochemical, and release properties of the formulations were determined as well as their potential for undesirable immunogenic reactions. Also, formulations composed of PLGA and BSA particles conjugated with transferrin (Tf) or rabies virus glycoprotein (RVG) were compared in order to select a formulation based on the criteria for a desired particle size and release profile. Of the four final formulations, the OT-loaded BSA nanoparticles conjugated to RVG were found to produce the smallest-sized particles and maintained the desired release profile of OT. In further embodiments, the composition comprised Tf-PLGA, Tf-BSA, RVG-PLGA, or RVG-BSA. These four formulations were formulated and characterized (see further details in the Examples hereinbelow). In one exemplary embodiment, a release profile provides an initial burst release of the active material (e.g., a peptide) followed by a sustained release. This is one exemplary profile for therapeutic effects, as well as the profile for in vivo testing. The data show that BSA particles conjugated to RVG offer useful properties.
Receptor-mediated transport (RMT) is a useful strategy for delivering drugs to the brain [17] and targeting ligands can be chosen or designed based on their ability interact with receptors on neural cells and transport a composition into the brain across the BBB. In one exemplary embodiment, transferrin (Tf) was coupled to the nanoparticles of the composition wherein the Tf was able to interact with the transferrin receptors expressed on the luminal membrane of capillary endothelium of the BBB [18-20]. In another exemplary embodiment RVG is coupled to the nanoparticles of the composition. Rabies virus glycoprotein (RVG) is a short peptide that can bind to nicotinic cholinergic receptors on the BBB and it has been employed as a targeting ligand to deliver small interfering RNA (siRNA) to the brain [48, 49]. In another exemplary embodiment, both Tf and RVG are coupled to the nanoparticles of the composition. In another exemplary embodiment the composition comprises two different groups of nanoparticles wherein the first group has Tf coupled to the first set of nanoparticles and the second group of nanoparticles has RVG coupled to the second set of nanoparticles. Both the first and second set comprise the composition. The first set may be administered first to a patient followed by the second set of nanoparticles. Alternatively, the first and second set of nanoparticles may be administered at the same time. With the provided new formulation and methods, compounds with potentially therapeutic use for CNS disorders that have been abandoned because of poor brain bioavailability can now be useful. Some of these compounds include neurotrophins such as BDNF [9, 10], secretase inhibitors for Alzheimer's disease [11], anti-inflammatory cytokines [12, 13], and therapeutic plasmid DNA for neuronal stimulation or myelin recovery and remodeling [14-16]. Neuropeptides such as neuropeptide Y, BDNF, orexin, and others have defined disease states where they show profound therapeutic promise, including epilepsy, substance-use disorders, and obesity.
The size, shape, and charge of the particles are important factors to consider for delivering drugs across the BBB [53]. It has been reported in a previous study that the negatively charged nanoparticles are highly endocytosed by cells due to their repulsive interactions with the negatively charged cell surface [54, 55], and positively charged particles show substantially reduce BBB penetrance. The nanoparticle formulations exhibit a smooth, regular and spherical shape, which helps maximize uptake, as spherical particles are efficiently taken up via both clathrin and caveolae mediated transport (see
To demonstrate the therapeutic viability of nanoparticulate formulations of certain exemplary embodiments of the present disclosure, both immunogenicity and cytotoxicity studies in vitro were completed. (See Examples 10 through 16 hereinbelow). As confirmed in past research, the nanoparticle formulations have shown to be both safe and non-toxic in non-human primates. In primates, all parameters related to liver toxicity (such as SGOT, SGPT) and kidney toxicity (such as serum creatinine, serum bilirubin) were normal after repeated nanoparticulate administrations when compared to the control group. Also, no antibody titers in response to the polymeric particle matrix were detected demonstrating the absence of an immune response to the polymer matrix formulation [36]. For confirmation of a potential innate inflammatory response, nitric oxide assays are an important tool. For the assay, dendritic cells are plated in a well plate and exposed to the formulation. After 16 hours, using reagents, a stronger color change indicates a higher production of nitric oxide by these antigen-presenting cells in response to the formulation's antigenicity. No significant difference in the amount of nitric oxide released in the supernatant of cells exposed to peptide-loaded nanoparticles compared to blank nanoparticles or the cells alone was demonstrated. This indicates minimal if not negligible immunogenicity of the formulation. For reference, nitric oxide release in response to the OT-loaded particles was substantially lower compared to cells that were exposed to a well-known highly-immunogenic breast cancer vaccine formulation. Likewise, cytotoxicity of blank nanoparticles was assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in a macrophage (RAW 246.7) cell line. No significant cell death following 48 hours of exposure to the formulations were found indicating that the formulation was not significantly toxic to the cells.
In one exemplary embodiment the nanoparticle formulation of compositions for peptide delivery protects the encapsulated peptide from degradation, and sustains the delivery of the therapeutic agent over an extended period of time. This sustained delivery allows for fewer administrations and easier compliance, an issue of particular importance in the treatment of substance abuse disorders for a prolonged amount of time or in cases where patients have difficulty with compliance. Resultantly, the active material (e.g., OT) can provide long-lasting therapeutic effects in the brain, improving the health of those suffering with various cognitive ailments.
In one exemplary embodiment, the compositions (also referred to herein as formulations) maintain a sustained release profile of OT in vitro in both PBS and plasma, which was confirmed using ELISA (
In the example described hereinabove, biodegradable Tf or RVG-conjugated PLGA and BSA nanoparticles loaded with oxytocin were successfully prepared by solvent evaporation and nanoprecipitation/coacervation methods respectively. The formulation was also characterized using various methods such as zeta sizing and scanning electron microscopy (See Examples). Significant parameters affecting particle size were ranked and optimized to produce the final formulations for in vivo testing. The nanoparticles produced were uniformly distributed in nanometer range, spherically shaped, and individually dispersed. The nanoparticles formulations were non-immunogenic and non-toxic. All four formulations showed sustained delivery by sustained release of the therapeutic agent allowing for fewer administrations and easier compliance. Therefore, the nanoparticulate formulation could serve as a potential candidate for delivering drugs and therapeutic peptides across the BBB non-invasively.
In one exemplary embodiment, a nanoparticle drug delivery system was developed and evaluated for OT designed to increase its brain bioavailability through active transport and sustain its delivery through encapsulation and sustained release. First, transport of OT-like large molecules using the technology in a cell culture model of the BBB was evaluated. Then, in vivo brain transport using bioimaging and cerebrospinal fluid (CSF) analysis was determined. Finally, the efficacy of OT in two different brain targeting and sustained-release formulations in a mouse model of social behavior was determined. One hypothesis is that this novel drug formulation increases the brain penetrance and sustains the delivery of intranasal OT.
Intranasal delivery is favorable when adverse or other effects limit oral dose delivery or when increasing brain penetrance is desirable. In one exemplary embodiment, the composition is provided to a patient intranasally. In this embodiment, the composition comprising an active material encapsulated by at least one nanoparticle and at least one targeting ligand coupled to the at least one nanoparticle, wherein the targeting ligand is chosen or designed because of its ability to interact with the luminal membrane of the BBB. The composition is administered intranasally to a patient wherein the composition crosses the BBB. In one exemplary embodiment, the composition is provided intranasally to treat a neurological disorder. In another embodiment the composition is provided in a liquid. The composition liquid mixture is aerosolized through a device to generate a mist of the composition liquid that is inhaled by a patient. The device can be any suitable aerosolizing device known to those skilled in the art.
In another exemplary embodiment, the composition comprises an active material, at least one nanoparticle to encapsulate the active compound, and a targeting ligand coupled to the at least one nanoparticle. The targeting ligand can be RVG. Further, the composition comprising the RVG targeting ligand is provided in a liquid. The liquid is aerosolized using a device known to those having ordinary skill in the art. The device aerosolizes the composition liquid creating a mist for a patient to inhale. The RVG targeting ligand interacts with nicotinic cholinergic receptors (nAChR) on the BBB to engage brain uptake of drug formulations (Kim et al. 2013; Liu et al. 2016). nAChR subunits are expressed in the nasal mucosa, including alpha and beta subunits (Keiger et al. 2003). There is evidence that nAChRs are expressed at significant densities on nasal trigeminal nerve endings of the rat, suggesting an anatomical distribution of these receptors that may facilitate direct bypass of the BBB (Alimohammadi and Silver 2000). Intranasal inoculation of the rabies virus is believed to allow the virus to directly access the brain via the olfactory epithelium, either along the olfactory nerve or the trigeminal nerve (Lafay et al. 1991; Terryn et al. 2014). In addition to RVG, there are a number of other receptor and transporter systems that allow for the development of novel brain targeting nanoparticle formulations. For example, the P-glycoprotein transporter, excitatory amino acid transporter, dopamine transporter, amino acid transporters, and nucleoside transporters among others are expressed in the nasal mucosa (Anand et al. 2014).
It is well known that OT alone is rapidly metabolized in both blood and cerebrospinal fluid (CSF). OT activates a plethora of systems in the periphery, most notably in the uterus, but in other organ systems as well. It is therefore important that intranasal OT should achieve the best possible ratio of brain to blood levels. Previous work in rodents, primates, and humans indicates that the doses required to achieve brain penetration may induce supraphysiological effects in the periphery (Born et al. 2002; Dal Monte et al. 2014; Leng and Ludwig 2016; Modi et al. 2014; Robertson et al. 1970; Striepens et al. 2013). This problem may not be addressable without a brain penetrance enhancer, as based on these studies, the calculated maximum transfer of a dose of OT to the brain within one hour of intranasal delivery is 0.005% (Leng and Ludwig 2016). Moreover, OT concentrations are basally higher in CSF than plasma, and the plasma spillover of intranasal OT thus drives a much higher proportional change in plasma than CSF. The composition and methods of use disclosed herein provide at least two significant advantages over intranasal delivery of OT alone. First, the release of the peptide is sustained and therefore likely limits the potential for repeated bolus activation of peripheral systems following the frequent dosing that would likely be required for intranasal OT in the absence of a sustained release formulation. Second, a greater proportion of the OT to the brain is targeted, both during the initial application and during any subsequent passes in the blood circulation that contacts the BBB, which increases the proportion in the brain relative to the periphery and may reduce the required dose. The in vitro and in vivo data support these contentions.
The transport of OT-like large molecules using the functionalized nanoparticle technology in a cell culture model of the BBB and in vivo using bioimaging was evaluated. The efficacy of OT in two different brain targeting and sustained-release formulations in a mouse model of social behavior was determined. It was found that the nanoparticle formulation of certain exemplary embodiments of the present disclosure increases BBB transport both in vitro and in vivo. Moreover, nanoparticle-encapsulated OT administered intranasally exhibited greater pro-social effects both acutely and 3 days after administration relative to OT alone. Both Tf and RVG nanoparticles produced robust acute and sustained elevations in overall social behavior that were comparable in their magnitude to each other. These acute pro-social effects were also in near agreement in their magnitude to those of MDMA in mice (Curry et al. 2018) and MDMA in rats (Morley et al. 2005; Thompson et al. 2007), which is a strongly prosocial and brain penetrant small molecule that elicits release of OT (Harris et al. 2002; Murnane et al. 2010; Murnane et al. 2012; Thompson et al. 2007). Interestingly, the two different brain targeting ligands differed in the nature of the pro-social effects that they elicited, with RVG selectively increasing general investigation and Tf increasing a mixed response of general investigation in some subjects and adjacent lying (data not shown) in other subjects with acute administration. Previous studies have shown that acute MDMA administration selectively increases general investigation in mice (Curry et al. 2018) but has more of a mixed response between general investigation and adjacent lying in rats (Morley et al. 2005; Thompson et al. 2007). It is possible that the differences between these two brain targeting ligands may be due to the kinetics of brain transport as mice that were treated with OT in nanoparticles conjugated to either ligand showed selective increases in general investigation 3 days after administration. These multimodal data strongly support the use of the approach to develop a brain targeting and sustained-release formulation of OT. This formulation can now be used to support intranasal delivery of OT and other neuropeptides.
The following examples are set forth for purposes of illustration only. Parts and percentages appearing in such examples are by weight unless otherwise stipulated.
Poly(D,L-lactide-co-glycolide) lactide:glycolide (50:50), polyvinyl alcohol, dichloromethane, indocyanine green, transferrin (Tf), and benzalkonium chloride were purchased from Sigma Aldrich (Milwaukee, Wis., USA). Bovine serum albumin (Fraction V), glutaraldehyde (25% in water) and acetone were purchased from Fischer Scientific (Pittsburgh, Pa., USA). Rabies Virus Glycoprotein (RVG) was purchased from GenScript (Piscataway, N.J., USA). All the chemicals were of analytical grade and used as is without further modification.
A full factorial design to optimize the parameters for the BSA nanoparticle formulation such as pH of the aqueous solution, polymer concentration, and antisolvent (acetone): solvent (water) ratio was performed. All three variables were evaluated at three different levels using a full factorial design of experiments. Results of the experiments were analyzed using JMP® (SAS) software as previously demonstrated by the group [37-39].
PLGA nanoparticles were prepared using a multiple emulsion solvent evaporation and lyophilization method. Briefly, 100 mg of PLGA and 20 μl of sorbitan monooleate were dissolved in 5 ml dichloromethane. 10 mg of OT was dissolved in 1 ml of deionized water. The water/oil emulsion was prepared by homogenization at 35,000 rpm for 1 minute and also followed by probe sonication for 5 minutes with a 30 second pause after each minute. This primary emulsion was added to 10 ml of a 1% w/v polyvinyl alcohol solution and homogenized using the same method as described for primary emulsion in order to obtain the final water/oil/water (w/o/w) emulsion. The emulsion was kept under stirring conditions for 6 hours to remove dichloromethane and the formulation was lyophilized and the dried particles were stored in glass vials at −20° C. for future use. BSA nanoparticles were prepared by a coacervation/nanoprecipitation method followed by lyophilization. Briefly, 270 mg of BSA and 27 mg of OT were dissolved in 10 ml of 10 mM NaCl at pH 9.3. Acetone was added drop wise at a speed of 1 ml/minute to the aqueous solution at a ratio of 2:1 with stirring at 800 rpm. After desolvation, the nanoparticle suspension was cross-linked for 30 minutes with 25% (w/v) glutaraldehyde in water (200 μL of glutaraldehyde for 1000 mg of BSA). Excess of glutaraldehyde was neutralized with sodium bisulphite. Trehalose (2% w/v) was added as a cryoprotectant and the nanoparticle suspension was lyophilized as above.
Previous studies have used Tf and RVG as brain targeting ligands [14, 18, 40] using other formulation technologies and to carry therapeutics other than neuropeptides into the brain. It was attempted to adopt these approaches for nanoparticle formulations of neuropeptides. Tf and RVG were conjugated to PLGA and BSA nanoparticles in two steps. The first step required activation of the carboxylic acid terminal groups using 1-ethyl-3-(3-dimethylaminopropyl) carbodimide (EDC) and NHS (98%) followed by conjugating Tf or RVG in the nanoparticles. Briefly, 1 ml (1 mg/ml) of EDC and NHS (1 ml, 1 mg/ml) was added to 10 ml of nanoparticle suspension containing 40 mg of OT-loaded PLGA or BSA nanoparticles. Carboxylic acid groups at the periphery were converted to amine-reactive esters by stirring the PLGA with EDC and sulfo-NHS reaction mixture at room temperature for 4 h. The activated NPs were then dispersed in 1 ml of PBS, and Tf (1 ml, 1 mg/ml) or RVG (1 ml, 1 mg/ml) was added drop-wise to the mixture. The mixture was stirred at room temperature for 2 h and incubated at 4° C. overnight or 12 hrs. Finally, the samples were washed and lyophilized using a Labconco freeze dryer (Labconco corporation, Missouri, USA). The 4 lead formulations were prepared as listed in Table 1
To determine the size and zeta potential of the nanoparticle formulations, a 50 μL aliquot of the nanoparticle suspension was diluted with 1.5 mL distilled water. Malvern Zetasizer (Malvern Instruments Inc., Massachusetts, USA) was used to measure the particle size and zeta potential of the nanoparticles utilizing the principle of dynamic light scattering. The results reported are average values from triplicate runs of three independent experiments for each sample. To visualize surface morphology, microparticles were mounted onto metal stubs using double-sided adhesive tape. After being vacuum coated with a thin layer)(100-150A° of gold, the microspheres were observed under 20 kV using a scanning electron microscope (Phenom World Pure Scanning electron microscope, AZ, USA) to examine the morphology of the particles.
To determine the encapsulation efficiency, a known amount of each nanoparticle suspension was centrifuged at a speed of 35,000 rpm. The supernatant was obtained and the amount of peptide in the supernatant was determined by an OT-specific enzyme linked immunosorbent assay (ELISA) (Oxytocin ELISA kit, Catalogue no: ADI-901-153A-0001, Lot no: 12041404E, Enzo life Sciences, NY 11735). This amount was subtracted from the total amount of peptide used for the formulation to determine the peptide encapsulation efficiency inside the nanoparticles.
The adsorption of Tf and RVG on the surface of PLGA and BSA nanoparticles was investigated using FT-IR spectroscopy in order to determine the conjugation efficiency. The FT-IR spectra were recorded using IRAffinity—1S Spectrometer (Shimadzu Corporation, Japan). FTIR spectra of transferrin and transferrin (Tf) and RVG coated PLGA and BSA nanoparticles were obtained in the range of 4000 to 600 cm−1. Spectral output was recorded by the transmittance as a function of wave number.
The potential of the formulations to induce an immunogenic reaction was assessed by measuring the amount of nitrite released by dendritic cells (DC) in presence of nanoparticulate formulations. Murine dendritic cells (DC 2.4, ATCC) were grown and maintained in DMEM with glucose, L-glutamine, and 10% FBS. DC cells were passaged at 70% confluence. For the assay, cells were plated in a 96 well-plate at 15,000 cells/well. The treatments consisted of untreated cells (negative control), the 4T1 immunogenic breast cancer vaccine (positive control), blank particles, and peptide particles. The cells were pulsed with 300 μg blank nanoparticles (1 mg/ml stock suspension) or peptide nanoparticles (1 mg/ml stock suspension) using incomplete DMEM in a 96-well plate for 16 hrs (n=3). After 16 hours, the supernatant was collected and analyzed for nitric oxide concentration using the Greiss chemical method. The Greiss reagent was prepared by mixing equal volumes of 1% sulfanilamide and 0.1% N-(1-napthylethyldiamine) solutions. One hundred microliters of the supernatant was transferred to a 96 well plate, to which 100 μl of Greiss reagent was added. The plate was incubated for 10 minutes and read at 540 nm to assess the color change using a microplate reader (Synergy H1; BIO-TEK Instruments, Winooski, Vt.). The concentration of nitrite was calculated for each well of cells using the standard curve of NaNO2 (1 mM stock concentration in distilled water further diluted to the highest standard at 100 μM followed by serial dilutions to 1.56 μM).
The cytotoxicity of blank and peptide loaded nanoparticles was assessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cell suspension equivalent to 10,000 cells/well was plated in a 96-well cell culture plate, and complete DMEM medium was added to obtain a volume of 100 μL per well. Various concentrations of the nanoparticles ranging from 0.5 μg/mL to 1000 μg/mL were added to the plates in triplicates and incubated for specified time points (24 and 48 hours). Cells alone (untreated) and benzalkonium chloride were used as negative and positive controls, respectively. 10 μL of MTT Reagent was added to each well, including controls followed by incubation at 37° C. for 2 to 4 hours. When the purple precipitate was clearly visible 100 μL of detergent reagent was added to all wells, including controls. Absorbance in each well was measured at 570 nm in a microtiter plate reader. The formation of a colored product quantified by measuring absorbance can be correlated to the percentage of living cells.
The release of OT from nanoparticles was studied in phosphate buffered saline (PBS, pH 7.4) and in 10% mice serum collected from Swiss-Webster mice by submandibular bleeding [41]. Briefly, 10 mg of nanoparticles were suspended in 1 mL of PBS or 10% mice serum and kept for continuous shaking (100 rpm). Two different temperatures were used, 4° C. and 37° C. At each time point, the tube was centrifuged at 35,000 rpm for 15 min, a sample of the supernatant was removed, after which the removed sample volume was replenished with media. The supernatant (n=3) was analyzed for the amount of peptide (OT) released using an OT ELISA as mentioned earlier.
Poly(D,L-lactide-co-glycolide) lactide:glycolide (50:50), Polyvinyl Alcohol, Dichloromethane and Indocyanine green (ICG) were purchased from Sigma Aldrich (Milwaukee, Wis., USA). Bovine serum albumin (Fraction V), glutaraldehyde (25% in water) and acetone were purchased from Fischer Scientific (Pittsburgh, Pa., USA). All the chemicals were of analytical grade and used as is without further modification. OT GenScript USA, Piscataway, N.J.) was dissolved in a mixture of 10% Tween-80 and saline and lightly vortexed for suspension. Nanoparticles equivalent to 50 μg of oxytocin was suspended in 50 μl of vehicle for each intranasal administration. This dose was based on preliminary experiments.
Bovine serum albumin (BSA) nanoparticles were prepared by a coacervation/nanoprecipitation method followed by lyophilization. Briefly, 270 mg of BSA and 27 mg of OT were dissolved in 10 ml of 10 mM NaCl at pH 9.3. Acetone was added drop wise at a speed of 1 ml/minute to the aqueous solution at a ratio of 2:1 with stirring at 800 rpm. After desolvation, the nanoparticle suspension was cross-linked for 30 minutes with 25% (w/v) glutaraldehyde in water (200 μL of glutaraldehyde for 1000 mg of BSA). Excess of glutaraldehyde was neutralized with sodium bisulphite. Trehalose (2% w/v) was added as a cryoprotectant and the nanoparticle suspension was lyophilized using Labconco freeze dryer (Labconco corporation, Missouri, USA).
Previous studies have used Tf and RVG as brain targeting ligands (Broadwell et al. 1996; Gan and Feng 2010; Kim et al. 2013; Liu et al. 2016; Pardridge 2014; Qian et al. 2002; Ulbrich et al. 2009) using other formulation technologies and to carry therapeutics other than neuropeptides into the brain. It was attempted to adopt their approaches for nanoparticle formulations of neuropeptides. Tf and RVG were conjugated to BSA nanoparticles in two steps. The first step required activation of the carboxylic acid terminal groups using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and NHS (98%) followed by conjugating Tf or RVG in the nanoparticles. Briefly, 1 ml (1 mg/ml) of EDC and NHS (1 ml, 1 mg/ml) was added to 10 ml of nanoparticle suspension containing 40 mg of OT-loaded BSA nanoparticles. The nanoparticles were dispersed in 1 ml of PBS, and Tf (1 ml, 1 mg/ml) or RVG (1 ml, 1 mg/ml) was added drop-wise to the mixture. The mixture was stirred at room temperature for 2 h and incubated at 4° C. overnight or 12 hrs. The samples were washed and lyophilized.
To generate a cellular model of the BBB, BEND3 cells were obtained from American Type Culture Collection (ATCC: 2299). BEND3 cells are endothelial cells that form tight barriers through the formation of transcellular tight junctions (Montesano et al. 1990; Sikorski et al. 1993; Williams et al. 1988), and these barriers mimic the barrier properties of the BBB (He et al. 2010; Watanabe et al. 2013). Their endothelial nature has been confirmed by the expression of von Willebrand factor and the uptake of fluorescently labeled low density lipoprotein. BEND3 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 15% FBS and 1% (100 U/ml Penicillin and 100 μg/ml Streptomycin). The cells were quickly thawed and centrifuged at 250×g for 8 minutes in 5 ml of complete media. The cell pellets were resuspended in 1 ml of complete media and plated on T-25 flask. Cells were maintained at 5% CO2, 37° C., and 80% humidity in a cell-culture incubator. At confluence, the cells were washed twice with 2.5 ml DPBS, trypsinized with TrypLE and centrifuged at 250×g for 8 minutes at 4° C. The cells were then resuspended in 1 ml of complete media and plated onto T-75 flask at appropriate densities for continuous passaging of the cells.
To characterize the membrane properties of the artificial BBB formed by these cells, the cells were seeded and cultured on permeable transwells using procedures optimized following the literature (Wuest et al. 2013). Confluent cells from T-75 flasks the cells were washed twice with 4 ml DPBS, trypsinized with TrypLE and centrifuged at 250×g for 8 minutes at 4° C. The cells were then resuspended in 1 ml of complete media. 150 of cell suspension was diluted with 150 of 0.4 trypan blue solution and a cell count was taken using a Countess II automated cell counting machine (MAN 0014293, Life Technologies, CA). The cells were diluted and seeded onto Costar permeable 12-well collagen coated membrane transwells with a 3 μm pore size (Corning; Corning, N.Y.) a density of 4×106 cells/insert in 0.5 ml of complete media. 1.5 ml of complete media was put on the basolateral side of the transwell while 0.5 ml of cell suspension was put into the apical side of the transwell insert. The media on both apical and basolateral sides of the transwell was changed every other day with complete media. All cells used for this work were below passage 33.
TEER measurements are a precise and well accepted method for assessing the barrier function of in vitro membranes prior to drug or formulation transport or barrier integrity studies (Li et al. 2004; Srinivasan et al. 2015). Following plating of the BEND3 cell on the transwells, the confluence of the cellular monolayer was manually monitored daily using microscopy. Furthermore, the resistance of the cellular monolayer was manually monitored daily using a Millicell-ERS-1 Epithelial Volt/Ohm Meter (EVOM Meter: MERS00001; MilliporeSigma; Burlington, Mass.). Briefly, the electrodes are sterilized with 70% ethanol and equilibrated in complete growth media. The resistance of the blank membrane was recorded as well as that of the cells, and the resistance of the blank membrane was subtracted from the resistance of the cell barrier. During resistance measurements, the electrodes are positioned such that the silver-silver chloride pellet neither touches the membrane of the insert nor is exposed to air. Actual voltage readings are obtained by subtracting blank resistance values. The resistance of cells was measured daily using the EVOM meter until resistance ranging from 50 to 100 ohms cm2 or more was obtained. This typically occurred between 6 to 8 days following plating.
To study the transport of nanoparticle formulations across the BEND3 BBB model barrier, a complete Ussing chamber system with a multichannel voltage-current clamp and the EasyMount Ussing chambers (Model VCC MC8; P2300; Physiologic Instruments Inc.; San Diego, Calif.) was used. The combination of the Ussing chambers and multichannel voltage-current clamp provided an apparatus for determining accurate nanoparticle transport and precise real-time measurements of the BEND3 membrane barrier function and bioelectric properties—TEER, short circuit current Isc, and potential difference PD. This system was used for final experiments once the cultures reached appropriate resistance as verified by EVOM measurements. To subtract the background resistance, a blank cell-culture insert was placed onto the slider and used to compensate for any fluid or blank membrane resistance by dialing off any reading to zero. Voltage differences between the electrodes were also offset. The blank inserts were then replaced with inserts containing cells and kept a temperature of 37° C. throughout the duration of the experiment. The cells were allowed to equilibrate in the Us sing chamber for 15 minutes before start of the experiment. Before the start of the experiment, all cell barrier preparations were evaluated for resistance properties and current flow using the acquire and analyze software. The resistance values of the cells during the experiment were obtained using the same software. For each experimental set up, 3 chambers contained cells while one was a blank insert.
For visual confirmation of monolayer formation, a solution (0.4%) of the visible dye trypan blue was used. This dye was chosen because its molecular weight (986.41 g/mol) is comparable to the molecular weight of OT (1006 g/mol). 100 μl of dye solution was put in the left side of each chamber and sampled from the right compartment at times 1, 2, 3 and 4 hours following dye administration. The intensity of dye at the various time points were quantified using a BioTek HT synergy spectrophotometer at 540 nm (Model S1AFR BioTek; Winooski, Vt.). To detect transport of the nanoparticles across the BBB, fluorescein isothiocyanate (FITC) was encapsulated in BSA nanoparticles and used to characterize transport of Tf-coated nanoparticles across the formed monolayer. 200 μl of nanoparticles solution of concentration 134 ug/ml solution was put in the left side of each chamber and sampled from the right compartment at times 1, 2, 3 and 4 hours. The fluorescent intensity of FITC at the various time points was quantified using Biotek fluorescent spectrophotometer at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The experiment was conducted in a dark environment to prevent fluorescent photobleaching of FITC.
Male Swiss-Webster mice (CFW; Charles River Laboratories, Inc.; Wilmington, Mass.) weighing between 25 and 40 g served as the subjects of the in vivo experiments. The mice were housed in groups of 4 in a temperature and humidity controlled room. Animals had access to food (Laboratory Rodent Diet) and water ad libitum. Mice were housed in rooms maintained in a 12-hour light/dark cycle. The lights were turned off at 6 pm every evening, and turned back on at 6 am every morning. All animals employed in this study were treated according to protocols evaluated and approved by Institutional Animal Care and Use Committee of Mercer University.
On experimental days, animals were administered ICG-loaded nanoparticles (6.7 ug of dye equivalent) intranasally, anaesthetized with isoflurane, and imaged using a LI-COR Odyssey Bioimager equipped with a MousePOD for in vivo imaging (LI-COR Biosciences; Lincoln, Nebr.). All mice were imaged at times 0.25, 1.5, 3, 6 and 24 hours. During imaging, animals were kept under anesthesia with isoflurane. The integrated intensity of ICG dye in the brain at 800 nm was quantified by the bioimager. For both Tf and RVG nanoparticles, more than 90% of the dye was cleared from the brain in 24 hours, most likely due to degradation of the dye itself rather than clearance of the nanoparticles from the brain. As the peak intensity of ICG dye in the brain was at approximately 2 hours for both Tf and RVG nanoparticles, the 2 hour time point for the subsequent in vivo cerebrospinal fluid (CSF) analysis and acute social behavior experiments were used.
Borosilicate glass capillary with filament (BD 100-75-10, Sutter Instrument Inc) was pulled in the middle over a Bunsen burner. The tip of the flamed glass capillary tubes was trimmed with scissors so that the tapered tip has an inner diameter of approximately 0.5 mm.
On sample collection days, animals were administered a 50 μl solution of OT alone or encapsulated in nanoparticles (containing 50 ug of OT) intranasally and placed in separate cages for 120 minutes.
CSF samples were collected from the cisterna magna as previously published by (Liu and Duff 2008) with little modifications. Briefly, the mice were anaesthetized with isoflurane and maintained under anesthesia at a flow rate of 1.5 ml/min during the surgical process. The mice were then placed prone in the stereotaxic instrument in direct contact with a heating pad with their heads firmly secured. The skin on the head and neck was shaved and the surgical site wiped with 10% povidone iodine followed by 70% ethanol. An incision was made and the muscles under the skin separated by blunt dissection under a dissecting microscope until the dura of the cisterna magna were exposed (a glistening white reverse triangle). The mice were then repositioned so the head formed a near 135° angle to the body. The dura mater was blotted dry with sterile cotton swab and saline. A capillary tube was inserted into the cisterna magna through the dura mater to allow CSF to flow into the glass capillary tube. The CSF was transferred into a pre-marked 0.6 ml Eppendorf, immediately placed on ice, and transferred into a −20° C. freezer. Any sample contaminated with blood (pink tinge) was discarded.
Immediately after CSF collection, blood samples were collected via the facial vein for each mouse into K2EDTA tubes (BD Microtainer 365974) and immediately placed on ice. The mouse was immediately euthanized by carbon dioxide asphyxiation. Blood samples were spun at 4200 rpm for 10 minutes at 4° C. after which the plasma fraction was collected and stored at −20° C. until analysis. Samples were not stored for more than 10 days.
All collected CSF and Plasma samples were treated identically and assayed at the same time with the same batch of reagents. All samples were tested using commercially available oxytocin ELISA kit (ADI-900-153A, Enzo Life Sciences, NY). The assay was performed according to the protocol provided by the manufacturer. Briefly, seven standards and samples were diluted in assay buffer and put in appropriate wells. To ensure ELISA kit was working appropriately, quality control metrics such as the total activity (TA), nonspecific binding (NSB), maximum binding (BO) and blank wells were included in the assay as recommended in the assay protocol. After addition of yellow antibody to all wells except for blank, TA and NSB wells, the plate was tapped gently, sealed, and incubated at 4° C. for 20 hours. After three washes, blue conjugate was added to TA wells and pNpp substrate solution was immediately added to all wells and incubated at room temperature without shaking for 1 hour after which stop solution was added. The optical density was read immediately at 405 nm with correction at 570 nm using BioTek HT synergy spectrophotometer at 540 nm (Model S1AFR BioTek; Winooski, Vt.). The mean optical density of the blank wells was subtracted from all readings.
Social-behavior experiments consisted of dyadic social interactions between familiar mice (mice housed in the same home cage). All experiments were conducted in boxes (33×25×9 cm) large enough to provide for social separation, with ambient lighting dim enough to allow interactions but bright to enable scoring of the videos. The bedding used for all experiments was from the home cages of the animals to prevent novelty-induced hyper-excitability. Experiments were conducted with the room kept at a minimal noise level. All videos were scored by a trained observer. On experimental days, animals were administered a 50 μl suspension of OT alone or encapsulated in nanoparticles (containing 50 μg of OT) intranasally and placed in separate cages for 120 minutes (n=4 to 6 pairs per group). The mice were then placed in the testing box, allowed to acclimatize in the box for 5 minutes and video recorded for 10 minutes. Behavior was rated on a 6-point scale, composed of: adjacent lying, anogenital sniffing, general investigation, body crossing, aggression, and no social interaction, as has been done previously (Curry et al. 2018).
All graphical data presentations and analyses were completed using GraphPad Prism (GraphPad Software Inc., La Jolla, Calif., USA). The cell culture data were analyzed by two-way analysis of variance (ANOVA). Bonferroni post-hoc analyses test was utilized to maintain a 95% confidence interval. The in vivo bioimaging data were analyzed by one-way ANOVA followed by Dunnett's post-hoc test. CSF concentrations of OT were assessed by unpaired t-test. The in vivo social behavior data were analyzed by one-way ANOVA followed by Tukey's post-hoc test. Post-hoc tested corrected for multiple planned comparisons to maintain the probability of making a type 1 error at 5%.
In general, the smaller the size of the nanoparticles, the more amenable they are for active transport across the BBB [42-45]. The mean diameter of PLGA nanoparticles measured by Malvern zeta-sizer ranged between 197.7 and 278.3 nm. PLGA-based particles were negatively charged ranging from −11.9 to −19.6 mV. The mean diameter of the BSA nanoparticles ranged from 100.1 to 197 nm in diameter. All the BSA nanoparticles were negatively charged ranging from −15.4 to −22.8 mV. The lowest particle size (approx. 100 nm) was obtained using a BSA formulation. Size distribution of RVG-conjugated OT-loaded BSA nanoparticles showed two peaks where intensity of the main peak was 99.24%. This may happen due to aggregation of some nanoparticles. As more than 99% particles were within a very narrow distribution range, it can be said that very few particles had aggregated. For Tf-conjugated OT-loaded BSA nanoparticles, the intensity of the main peak was 92.6%. For RVG- and TF-conjugated OT-loaded PLGA nanoparticles there was only one peak for each type of particles and the intensities of the peaks were 99.9% and 100% respectively.
Parameters affecting the particle size are rank ordered by analysis of the formulation design with JMP® (SAS) software. Significant parameters (p<0.05) affecting particle size (in the order of importance) are interaction of BSA concentration and acetone: water ratio (p<0.0005) pH of the aqueous solution (p<0.0006), interaction of pH and acetone: water ratio (p<0.0013), acetone: water ratio (p<0.0053). The optimization is listed in Table 3.
Transferrin (Tf) and Rabies Virus Glycoprotein (RVG) Conjugation to Peptide Loaded Nanoparticles
The encapsulation efficiency was determined before the conjugation of the nanoparticles to Tf and RVG. The amount of peptide in the supernatant was subtracted from the total amount of peptide used for the formulation to determine the peptide encapsulation efficiency inside the nanoparticles. The oxytocin peptide content of the PLGA nanoparticles was 81±2.5% (w/w) and for BSA nanoparticles it was 75±2.5% (w/w).
Nitric oxide release is an important indicator of innate immune response [46,47]. This can be used to determine the potential of nanoparticle formulations to induce immunogenic reactions.
The cytotoxic effects of the four lead nanoparticle formulations (as mentioned in Table 1) at given concentrations are depicted in
The release of OT from PLGA nanoparticles in PBS was sustained over a period of 21 days (
The integrity of the artificial BBB barrier was assessed using trypan blue transport. The resistance of cellular barrier (BEND3 cells) at first sampling point, t=60 min was Rt 123.7+54 Ω·cm2 n=3 and at t=240 min was Rt 117.2+42.5 Ω·cm2; n=3. Resistance without a cellular barrier, t=60 min was Rt 2.89+1.2 Ω·cm2 n=3 and at t=240 min was Rt 4.2+2.7 Ω·cm2 n=15.
The transport of trypan blue across cell barrier was (
Transport of FITC in brain targeting nanoparticles across the artificial BBB barrier (
The time course of brain uptake of ICG alone or ICG in each of the two formulations was determined. The area under the curve (AUC) of the time course in each animal was determined and tabulated for statistical analyses. There was a significant main effect of treatment (F2,12=5.098; p<0.0250) on brain penetrance of ICG nanoparticles (
Consistent with the bioimaging data, 2 hours after intranasal administration, significantly higher levels of OT in CSF (t=7.501; p<0.05) when it was administered in RVG-conjugated nanoparticles compared to when it was administered alone (
One-way ANOVA revealed a significant main effect of treatment (F3,32=8.897; p<0.0002) on murine social interactions (
Although only a number of exemplary embodiments have been described in detail above, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages. Accordingly, all such modifications are intended to be included within the scope of this disclosure as defined in the following claims.
While the methods, equipment and systems have been described in connection with specific embodiments, it is not intended that the scope be limited to the particular embodiments set forth, as the embodiments herein are intended in all respects to be illustrative rather than restrictive.
Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect.
Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
“Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. “Exemplary” means “an example of” and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes.
Disclosed are components that can be used to perform the disclosed methods, equipment and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc., of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods, equipment and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
It should further be noted that all patents, applications, and publications referred to herein are incorporated by reference in their entirety.
Transcytosis of protein through the mammalian cerebral epithelium and endothelium. III. Receptor-mediated transcytosis through the blood-brain barrier of blood-borne transferrin and antibody against the transferrin receptor. Experimental neurology. 1996; 142:47-65.
Nanoparticle-mediated brain drug delivery: Overcoming blood-brain barrier to treat neurodegenerative diseases. Journal of controlled release: official journal of the Controlled Release Society. 2016; 235:34-47.
This application claims benefit of co-pending U.S. provisional patent application No. 62/696,875, filed Jul. 12, 2018, entitled NANOFORMULATIONS OF NEUROPEPTIDES AND METHOD OF MAKING SAME, and commonly assigned to the assignee of the present application, the disclosure of which is incorporated by reference in its entirety herein
This invention was made with government support under NS 100512 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62696875 | Jul 2018 | US |
