Method for preparing pH dependent ultra small polymeric nanoparticles for topical and/or transdermal delivery

Abstract

The invention provides a new method for preparing ultra-small polymeric-lipidic delivery nanoparticles (USDNs) that were synthesized by a nanoprecipitation method followed by a layer-by-layer nanodeposition. The USDNs particle size can be controlled between 5-25 nm and provides loading capacities of 22.12% to 72.08%. Moreover, the USDNs platform provides pH controlled drug release, within a terminal release ratio of 68% at pH 5.0 and almost no release to pH of 7.5. Furthermore, based on their small sizes (5-25 nm) and unique composition, the USDNs penetrates the skin strata efficiently, release the payload at the target site as topical or transdermal treatment of a variety of skin disorders. Additionally the USDNs system can be used to treat and diagnoses other crucial diseases (Cancer, Alzheimer, etc) can be combined with various micro-needles or needles free array technologies for special application.

Claims

1. A method for making a hybrid, biodegradable, non-toxic phospholipid polymer nanoparticle, comprising i) nanoprecipitating a biodegradable polymer, wherein the biodegradable polymer is selected from the group consisting of polylactic (PLA) and polvglycolic (PGA) polymers; poly lactic-co-glycolic acid copolymers (PLGA): diblock copolymers containing a functional poly(ethylene glycol) (PEG) and PLGA (PEG-PLGA); PEG-PLA diblocks: triblock copolymers containing PEG and PLGA; polymers and copolymers of polycaprolactones polymer; polycaprolactones-(poly(acrylic acid) (PAA) copolymer; (2-ethyl-2-oxazoline) (PEtOz) poly(N-isopropylacrylamide; poly(N,N-dimethylamino-2-ethyl methacrylate) linked to a hydrophobic polycaprolactones segment: poly(alkyl cyanoacrylates); poly(ortho esters); poly(anhydrides) of poly(sebacic acid), poly(adipic acid) or poly(terphthalic acid); poly(amides); polyester amides) and poly(phosphoesters) ii. stabilizing said biodegradable polymer with a phospholipid-polymer layer to produce a stabilized nanoprecipitate, iii) adding a natural polymer, wherein the natural polymer is a protein-based polymer or polysaccharide, iv) reacting said stabilized nanoprecipitate and said natural polymer, v) adding at least one active substance, wherein said at least one active substance is added: a) to the stabilized nanoprecipitate produced in step ii) before reacting said stabilized nanoprecipitate and said natural polymer, to encapsulate the active substance between a polymer shell and an outer shell comprising a natural polymer, and/or b) to the biodegradable polymer of step i) to produce a polymer-active substance mixture before stabilizing the polymer-active substance mixture with a phospholipid layer in step ii), which results in a lipid core encapsulated nanoprecipitate having at least one active substance entrapped inside the phospholipid core, vi) filtering any resulting nanoparticles, wherein said hybrid, biodegradable, non-toxic phospholipid-polymer nanoparticle, comprises a) a phospholipid core comprising a biodegradable polymer and a phospholipid, b) a polymer shell encapsulating the phospholipid core, c) an outer shell comprising a natural polymer, and d) at least one active substance which is encapsulated inside the phospholipid core, and/or between the polymer shell and the outer shell comprising the natural polymer, and wherein said hybrid, biodegradable, non-toxic lipid polymer nanoparticle is 3 nm-25 nm in size.

2. A method for increasing skin penetration of a topically or transdermally administered active substance, comprising encapsulating said active substance in the hybrid, biodegradable, non-toxic lipid-polymer nanoparticle made according to claim 1 and topically administering said hybrid, biodegradable, non-toxic lipid-polymer nanoparticle to a patient in need of such treatment.

3. The method according to claim 1, wherein the polylactic-co-glycolic acid polymers are selected from the group consisting of aliphatic polyesters PGA, PLA, and PLGA; the PLGA copolymer is a PLGA and PEG copolymer; and/or the poly(amides) are selected from the group consisting of poly(amino acids), poly(-glutamic acid) and poly(L-lysine).

4. The method according to claim 1, wherein said protein-based polymer is selected from the group consisting of collagen, albumin, and gelatin and/or said polysaccharide is selected from the group consisting of agarose, alginate, carrageenan, hyaluronic acid (HA), dextran, chitosan, and cyclodextrins.

5. The method according to claim 1, wherein at least two active substances are added, wherein at least one active substance is encapsulated in the lipid core and at least one active substance is encapsulated between the biodegradable polymer shell and the outer shell comprising a natural polymer shell, and wherein one active substance is hydrophobic and one active substance is hydrophilic.

6. The method according to claim 5, wherein the outer shell comprising the natural polymer further comprises groups for the conjugation of targeting bioactive molecules and/or delivery enhancers.

7. The method according to claim 6, wherein the targeting bioactive molecules are bio-affinitive ligands that recognize a specific cell.

8. The method according to claim 6, wherein the delivery enhancers are bio-affinitive cell penetrating peptides selected from the group consisting of a transactivator of transcription (TAT) protein from the HIV virus, meganin, penetratin, TD-1, SPACE-peptide, IMT-P8, antennapedia, transportan and polyarginine.

9. The method according to claim 1, wherein said active substance is selected from the group consisting of an anti-aging agent, an anti-wrinkle agent, a drug for treating skin disorders, a chemotherapeutic agent, a diagnostic agent, an antibiotic, an antiseptic agent and an antiviral agent.

10. The method according to claim 1, wherein the lipid core comprises carboxylic acid-terminated poly(lactic-co-glycolic acid) and 1,2-Distearoyl-snglycero-3-phosphoethanolamine, and the biodegradable polymer shell comprises polyethylene glycol and the natural polymer is chitosan.

11. A hybrid, biodegradable, non-toxic phospholipid-polymer nanoparticle, comprising a) a lipid core comprising a biodegradable polymer and a phospholipid, b) a polymer shell encapsulating the phospholipid core, c) an outer shell comprising a natural polymer, and d) at least one active substance which is encapsulated inside the lipid core, and/or between the polymer shell and the outer shell comprising the natural polymer, wherein said hybrid, biodegradable, non-toxic lipid-polymer nanoparticle is 3 nm-25 nm in size, and wherein said hybrid, biodegradable, non-toxic lipid-polymer nanoparticle is produced by a method comprising: i) nanoprecipitating a biodegradable polymer, wherein the polymer is selected from the group consisting of polylactic (PLA) and polyglycolic (PGA) polymers; poly lactic-co-glycolic acid copolymers (PLGA); diblock copolymers containing a functional poly(ethylene glycol) (PEG) and PLGA (PEG-PLGA); PEG-PLA diblocks; triblock copolymers containing PEG and PLGA; polymers and copolymers of polycaprolactones polymer; polycaprolactones-(poly(acrylic acid) (PAA) copolymer; (2-ethyl-2-oxazoline) (PEtOz); poly(N-isopropylacrylamid; poly(N,N-dimethylamino-2-ethyl methacrylate) linked to a hydrophobic polycaprolactones segment; poly(alkyl cyanoacrylates); poly(ortho esters); poly(anhydrides) of poly(sebacic acid), poly(adipic acid) or poly(terphthalic acid); poly(amides); polyester amides) and poly(phosphoesters) ii. stabilizing said biodegradable polymer with a phospholipid polymer layer to produce a stabilized nanoprecipitate, iii) adding a natural polymer, wherein the natural polymer is a protein-based polymer or polysaccharide, iv) reacting said stabilized nanoprecipitate and said natural polymer, v) adding at least one active substance, wherein said at least one active substance is added: a) to the stabilized nanoprecipitate produced in step ii) before reacting said stabilized nanoprecipitate and said natural polymer, to encapsulate the active substance between a polymer shell and an outer shell comprising a natural polymer, and/or b) to the biodegradable polymer of step i) to produce a polymer-active substance mixture before stabilizing the polymer-active substance mixture with a phospholipid layer in step ii), which results in a lipid core encapsulated nanoprecipitate having at least one active substance entrapped inside the lipid core, and vi) filtering any resulting nanoparticles.

Description

DESCRIPTION OF THE INVENTION

(1) In certain embodiments, this disclosure relates to a layer by layer ultra small hybrid biodegradable and non-toxic nanoparticle (USDNs) comprised of a lipid core, followed by a two subsequently shell of a middle biodegradable polymer layer entrapped in an outer natural polymer. These nanoparticles can be encapsulated with at least one active substance, wherein; the active substance can comprise but is not limited to the following group of therapeutic active substances: an anti-aging and anti-wrinkle agent, a drug or a group of drugs or active biological molecules with psoriasis and rosacea or any other skin disorder, a chemeotherapeutic agent, an antibiotic, anitispetic or antiviral agent, wherein, the said USDNs surface contain delivery enhancers molecules (TAT, IMT-P8, etc) and can be also conjugated with any bio-active ligand for targeted drug delivery, wherein said, the USDNs is used as a topical or/and transdermal delivery, wherein said, the USDNs penetrates the skin strata efficiently, release the active substances at the target site as topical or transdermal treatment for a variety of skin disorders, wherein, the USDNs system can be also used to treat and diagnoses other crucial diseases (Cancer, Alzheimer, etc) and can be combined with various micro-needles or needles-free array technologies for special application.

(2) Given the potential benefits of both the size and composition, the present invention relates a facile synthesis method of a biodegradable lipid-polymer nanoparticle having a size less then 25 nm, preferable less than 10 nm and most preferable of 5 nm. Active molecules can be inserted into the nanoparticles, including more than one active molecule such as therapeutic agents and diagnosis agents. The previously reported sizes of hybrid layer-by-layer nanoparticles were from 50 nm to grater than 200 nm.

(3) The particles has a hybrid polymer-lipid composition, where the lipid is from a class of phospholipids, wherein, the preferred lipid is 1,2-Distearoyl-snglycero-3-phosphoethanolamine and the polymer is from a class of biodegradable polymers and natural polymers, wherein, the preferred polymer is Carboxylic acid-terminated poly(lactic-co-glycolic acid) (PLGACOOH; 50:50 ratio) and chitosan. The USDNs is such designed to have enough solubility in the lipid domain of the stratum corneum, while still having sufficient hydrophilic nature to allow partitioning into the skin inner layers for an optimum delivery. The USDNs lipid core has a hydrophobic nature that enables to encapsulate and deliver high molecular weight or/and hydrophobic drugs .sup.37, which have poor penetration properties through the sin barrier. The lipid core is then surrounded in a biodegradable polymer shell (that wraps the core and co-encapsulates more drags) and subsequently covered by outer chitosan-TAT layer. Herein, the TAT peptide is used as a penetration enhancer, chitosan was used both to stabilize the hybrid platform and to co-encapsulate active molecules.

(4) The ultra small nanoparticles (5 nm) stability in water, phosphate buffer saline (PBS at pH-8), and under physiological conditions (in fetal bovine serum) was determined by DLS. The analysis was conducted for 24 hours and slight change of the hydrodynamic size were observed suggesting that presence of highly strength media and the physiological condition do not change the surface properties of the nanoparticle significantly. The system such made is stable in relevant physiological conditions and can co-encapsulate higher quantities of active substance, biological molecules, etc.

(5) The co-encapsulation of multiple active molecules is processed as follows: (1) into the core and (2) onto the lipid layer (between the lipid layer and the chitosan shell) into one platform, wherein, the desired intrinsic properties and integrity of the platform and of the active compounds present into the platform is not affected. The USDNs platform and the encapsulated molecules offer independent and associative properties to each individual component of die system. Moreover, the loading of the drugs has little effect of the size and disparity of the USDNs.

(6) Drugs@USDNs appears d with similar average particle size and the encapsulated drugs are related to the psoriasis treatment such as: (1) efalizumab; and (2) the interleukin-12/23 (IL-12/23) monoclonal antibody, ustekinumab, where, the CD11a chain of LFA-1 inhibitor and cell adhesion, efalizumab, was encapsulated in the core and the antibody between the shells. The zeta potential of the Drugs@USDNs was 4.2 mV, which increased to 12.6 and 15.0 mV after surface modification with TAT. The presence of the TAT amino groups endowed the Drugs@USDNs platform with positive charge.

(7) The antibody, ustekinumab is presently used as an injectable and require repeated injections and some patients experience a loss of therapeutic effect. Moreover, efalizumab was withdrawn from the market because of serious side effects. Therefore, using the present invention platform co-encapsulation, the side effects and the toxicity was highly diminished or overcome. Moreover, the system allows lower quantity of drugs to be delivered, thus, resulting in lower or even zero toxicity and high delivery and treatment performance.

(8) Drug Loading and pH-Mediated Release:

(9) Two important factors that determine the efficiency of the Nanocarrier are the loading capacity and the release behavior. The drugs loaded onto the platform are two drugs with activity in the treatment of psoriasis: efalizumab and ustekinumab where, the CM11a chain of LFA-1 inhibitor and cell adhesion, efalizumab was encapsulated in the core and the antibody, ustekinumab, between the core and chitosan shells.

(10) The co-delivery platform, Drugs@USDNs of 5 nm and respectively 10 nm, has a drug loading capacity of about 300-500 /mg but most preferable of 400 g/mg. Moreover, since the release behavior of the platform is pH-mediated, the platform was exposed in PBS under various pH conditions varying from pH=[7.4-5.0]. The pH-mediated release has an important impact on the platform's application. In various skin diseases, the pH of the stratum corneum and epidermis, dermis, etc is deregulated by the activity of the pH dependent enzymes, which regulates skin cornification desquamation and homeostasis of the barrier function. In patients suffering from epidermal photogene's such psoriasis, the skin surface pH has been reported to be between pH=[6-6.5]. .sup.38 Therefore, the herein platform, under acidic conditions (pH=5.5-6.5) is leaking out the drugs with a 38.65% and 27.81% release ratio in 12 h, and a terminal release ratio of was determined to be at 60.91% and respectively, 52.14% in 90 h. However, under neutrally condition (pH 7.4), almost no drugs were released (4.2%). The chitosan layer is responsible for the first release of the drug, the denaturation of its amino acids in acidic conditions leads to the destabilization of the layer and the release of the drug, while, the lipid core degrade as well in strong acidic to lower acidic condition.

(11) In conclusion, the patent relates a new method to synthesize an ultra small hybrid platform that has the ability to introduce targeting functionalities on its surface as well as co-encapsulate two or more active ingredients and release them to the targeted sites in a percentages within 22.12% to 72.08% at pH 5.0 and almost no release to pH of 7.4 or up. Due to its size and composition, this platform has a wide range of applications, including topical and transdermal delivery, brain targeted delivery, tumor penetration, vaccination for the treatment of various diseases.

(12) In Vitro Delivery of Drugs@USDNs:

(13) Herein, the disclosed fabrication methods of the USDNs provide the ability to functionalize and stabilize its surface, and provide tailored characteristics that improve the delivery and treatment/diagnosis. Many active ingredients can be encapsulated into the nanoparticles and delivered to the payload site. An active ingredient can be a substance that is administrated into the body especially for a desired application, and has a biological effect on the organism. The USDNs can be loaded with both hydrophilic and hydrophobic active ingredients.

(14) Based on its small sizes and composition, the USDNs have a high capacity to carry active molecules into the cells. Moreover, the internalization is enhanced by the presence of TAT molecules on its surface. The internalization efficiency was monitored in-vitro by incubating the platform with meduloblastoma cells and using Using florescence microscopy to confirm its internalization. In a typically study, 5 M of Drugs@USDNs was functionalized with FITC and then incubated with meduloblastoma cells for 1 h at 37 C. The uptake of the Drugs@USDNs-FITC (contain TAT) was compared with Drugs@USDNs-FITC (without TAT). Considerable higher florescence was observed for the when cells were treated with Drugs@USDNs-FITC (contain TAT), which indicates that TAT increases the internalization capacity of the platform.

(15) Skin Penetrating Properties of LMT-P8 In Vivo:

(16) Given the results demonstrating that Drugs@USDNs is capable of transporting the medication inside the cells, a further investigation of the ability to penetrate the stratum corneum and enter into epidermis was performed; so that, it could be used as carrier for topical and/or transdermal drug delivery. After 2 h the skin section treated with FITC-Drugs@USDNs, the green florescence was observed in epidermis with diffusion in the dermal tissue, which suggests the ability of the platform to penetrate the stratum corenum efficiently and accumulate into the epidermis in a short period of time. Additionally, lower green florescence was observed in the hair follicles.

EXPERIMENTS

(17) Synthesis of the pH Dependent Ultra Small Polymeric Nanoparticles (Drugs@USDNs).

(18) Disolution of Chitosan 1% in HCl Acid

(19) Chitosan solution (0.1%) was prepared by dissolving 1 g of chitosan in 90 ml of distilled water containing 20 in of HCl and by mixing the followed solution at 60 C. for 24 h.

(20) Preparation of TAT-Chitosan

(21) First, TAT-chitosan conjugation was performed as follows: 1 ml of 1% chitosan in 1% HCl solution containing by adjusting the pH-6 were mixed with 200 mM EDAC and 200 mM NHSS at room temperature for 5 min. After, 5 mg of HIV-1 Tat peptide was added to the chitosan solution and stirred for 4 h at room temperature. Next, 1 mg of tris(2-carboxy-ethyl)phosphine hydrochloride was added and the solution was then left undisturbed for 30 min. The sample was then purified using centrifugation.

(22) Layer-by-Layer Synthesis of the USDNs

(23) The preparation of the USDNs was preformed using the following nanoprecipitation process such as: Step 1. At pH=8, the carboxylic acid-terminated polylactic-co-glycolic acid) (1 mg mL-1), previously dissolved in acetonitrile, was precipitated and stabilized with a solution of 1,2-Distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] dissolved in Tris-HCl. Step.2. Further, 2 ml TAT-chitosan was added into the solution and the reaction was continued for another 8 h. The resulting nanoparticles were filtrated using a 100 kDa Amicon centrifugal filter and the size were measure by Transmission electron microscopy (TEM). The TEM grids were prepared by depositing a 30 l of the as prepared particles on a 100 meshes cupper grid. The resulted nanoparticle have a size of 5 nm. In order to obtain larger sizes of nanoparticles, of 5-25 nm, the concentration of TAT-chitosan was vary between 1-5 ml, wherein the most preferable are ultra small sizes of 5 nm.

(24) The nanoparticles are highly stabile both in psychological conditions and water and their stability was analyzed in: phosphate buffer saline (PBS), fetal bovine serum (FBS) and 10 mM Tris-HCl a: pH 8 for 2 weeks. Such as, 2 mg ml.sup.1 of nanoparticles were added to 2 ml of PBS/FBS/Water and DLS measurements were performed twice a day until day 14.

(25) Drugs or Active Substances Loading into the USDNs Platform

(26) The CD11a chain of LFA-1 inhibitor and cell adhesion, efalizumab, and the interleukin-12/23 (IL-12/23) monoclonal antibody, ustekinumab, were loaded into the USDNs platform by physically encapsulation. The efalizumab was dissolved into the PLGA phase and the synthesis was processed as described above at Step.1. Further, ustekinumab was added into the nanoprecipitate and then Step 2 of the reaction was followed. (as described above at Layer-by-Layer Synthesis of the USDNs). Then, thw resulted nanoparticles were purified by using a 100 KDa MWCO Amicon centrifugal filters to remove the excess of the encapsulated drugs and other unreacted row materials. The quantity of the drug loaded was quantified by using PerkinElmer Series 200 high performance liquid chromatography (HPLC) system using a C18 analytical column (Brownlee) with a mobile phase of 50:50 water to acetonitrile and a detection wavelength of 230 nm. Moreover, the quantity of drug released and retained at various pH and time was assessed.

(27) Toxicity of Nanoparticles USDNs Platform

(28) Cytotoxicity of the nanoparticles was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-iphenyltetrazolium bromide (MTT) assay with two different cell lines, i.e., HEK293 human embryonic kidney cell, HeLa and D556human brain tumor medulloblastoma cell. The cells were maintained as an adherent culture and grown as a monolayer in a humidified incubator (95% air, 5% CO2) at 37 C. in a cell culture flask containing medium supplemented with 1% penicillin-streptomycin and 10% FBS. Cells were detached and seeded in 96-well flat-bottom microplates at 4000 cells per well. After 24 h recovery at 37 C., the medium was replaced with 100 l medium containing nanoparticles (USDNs or Drug@USDNs) at various concentrations (12.5-400 g/ml). For the control cell sample, a fresh medium without nanoparticles was added. After 24 h of incubation at 37 C., 10 l of MTT solution (5 mg/ml) was added into each well following a 4 h incubation period. After removing the culture media, the precipitated formazan was then dissolved in 10% SDS in 0.01M HCl. Finally, a micro plate reader (Biotech Synergy2) was used to measure the absorption of all samples (n=6 per group) at 570 nm. Cell viability was determined by comparing the absorptions of cells incubated with and those without nanoparticles.

CITATION LIST

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