Description:ELASTIC NANO-VESICLES FOR CO-DELIVERY OF ANTIGLAUCOMA AGENT(S) AND SIRNA FOR TREATMENT OF GLAUCOMA AND METHOD OF PREPARATION THEREOF
TECHNICAL FIELD
The present invention relates dendrimeric peptides (DPs), Hyaluronic acid (HA) and its formulation for enhanced permeability/ targeted delivery of active agent(s). The invention is specifically related to the use of dendrimeric peptide attached to nano-vesicles (NV) loaded with active agent(s) for enhancing the corneal permeability of the formulation and ii) active agent(s) loaded nano-vesicles coated with targeting ligand (HA) for improving the targeting efficiency into the posterior segments of the eye. The present invention particularly relates to elastic nano-vesicles for co-delivery of antiglaucoma agents and siRNA for treatment of glaucoma and method of preparing the same.
BACKGROUND OF THE INVENTION
Glaucoma is an eye disease in which intraocular pressure (IOP) rises above 21 mm Hg (normal IOP 15-20 mm Hg) and if it is left untreated, the patient’s vision may be lost (Consoli & Ramlogan, 2015; Lee & Higginbotham, 2005). The main challenge in ocular drug delivery is to circumvent the eye’s protective barriers so that the medication ventures into bio-milieu in adequate concentration to treat ocular anomalies (Kaul et al., 2012; Wadhwa et al., 2009)).
Recent advances in drug delivery and biomaterial engineering have led to the development of nanotechnology-based drug delivery systems to precisely deliver the drug at the site of action and hence circumvent the failures associated with conventional therapy of glaucoma. Nanotechnology-based treatment strategies have now been routinely applied in the treatment of ocular disorders affecting the anterior and posterior segments of the human eye (Weng et al., 2017). Novel ocular drug delivery has been explored to circumvent the limitations associated with conventional ocular drug delivery systems.
Nano formulations enhance the ocular bioavailability by increasing the precorneal residence time of the drug and decreasing the partial loss of the drug after instillation. Most antiglaucoma drugs have low corneal permeability and thus low bioavailability. Novel ocular drug delivery systems enable the drug targeting to the posterior segments of the eye by increasing the corneal permeation, reducing the dosing frequency, and reducing drug-related side effects and toxicities. Other benefits of nanotechnology-based ocular drug delivery systems include ease of self-administration, lack of blur vision, resistance to metabolic enzymes, and potential corneal cell uptake. Various nanotechnology-based drug delivery systems such as liposomes, nanoparticles, microemulsion, nanosuspension, micelles, hydrogels, dendrimers, niosomes, cubosomes, spanlastics, nanofibers, etc., have been developed (Wadhwa et al., 2009; Wang et al., 2018).
Dendrimers are artificial macromolecules with characteristic globular shapes, highly branched points, mono-dispersity, and size range in nano-metres. To prolong the drug’s residence time on the ocular surface and decrease systemic absorption, the usage of dendritic structures has recently been investigated. Many reports are available on the delivery of drugs as well as siRNA safely and effectively at the specific target site using dendrimers (Hegde et al., 2017; Luo et al., 2011; Mutalik et al., 2009). The previous studies conducted on PAMAM dendrimers discovered that the corneal residence time and the delivery efficacy of the drug were enhanced for dendrimers. In general, these assays revealed enhanced corneal residence time and drug delivery efficacy for bigger and hydroxyl-terminated dendrimers. The authors hypothesized that this effect was mostly caused by dendrimers' interaction with ocular mucins, which prevented the drug washout (Vandamme & Brobeck, 2005; Yao et al., 2010). The ex vivo corneal permeation experiments showed that with the increasing generation of dendrimer the efficacy of drug delivery improved considerably due to the disruption of the tight epithelial junctions of the corneal membrane by the cationic dendrimer particles (Bravo-Osuna et al., 2012; Grimaudo et al., 2017; Souza et al., 2015).
Negatively charged biological membranes are known to interact with cationic dendrimers and become more permeable. Because the cornea is negatively charged at physiological pH levels, cationic dendrimer interaction with the cornea is anticipated (Hong et al., 2004; Venuganti & Perumal, 2008). The dendrimers induce the formation of a hole in the lipid bilayer and loosen the epithelial cell junctions which change the physical properties of the cell membrane leading to the solubilization of the corneal membrane (Yao et al., 2010). Since peptide dendrimers (DP) (also known as dendrimeric peptides or amino acid-based dendrimers) are non-toxic, economical, biodegradable, and easy to purify. They are used as an alternative to other types of dendrimers, such as PAMAM dendrimers, in the present study (Manikkath et al., 2016; Mutalik et al., 2014). Solid phase peptide synthesis (SPPS) method and liquid phase peptide synthesis (LPPS) methods are generally used in the synthesis of dendrimeric peptides which involves the sequential addition of amino acid chains on to an inert resin substrate. SPPS method is more widely used in the synthesis of DP in comparison to LPPS method as it is more efficient and economical, faster reaction kinetics, no isolation and characterization of the intermediates, and prevention of the racemization of the amino acids (Fischer et al., 2014).
Ligand mediated drug delivery has gained much attention over the years because exploiting natural ligands and their receptors for site-specific delivery results in enhanced therapeutic index besides their advantage of being biocompatible, non-immunogenic and biodegradable. HA is a natural component distributed widely in various ocular tissues such as the cornea, aqueous humour, iris, lens, vitreous humour, and retinal cells (retinal pigment epithelial cells, muller cells, and retinal ganglion cells) and thus has attracted more attention in ocular drug delivery systems (Guter & Breunig, 2017). Because of its interaction with the precorneal mucin layer through non-covalent bonding, HA prolongs the time that the drug stays on the ocular surface, decreases drug loss, and increases drug bioavailability (Zeng et al., 2016). Another goal of the current study was to coat the siRNA-loaded nano-vesicles with retinal ganglion cell specific ligand like HA to knock down the overly expressed caspase 2 genes in glaucoma patients.
In the publication Manikkath et al., 2017; Mutalik et al., 2012; Shetty et al., 2017, the inventors discussed the potential of dendrimeric peptides as permeation enhancers for transdermal drug delivery. However, no prior art teaching the conjugation of DP in the Timolol maleate (TM) loaded nanocarrier/ nano-vesicle system to improve the corneal permeability of the elastic nano-vesicles.
Based on the foregoing, it is believed that a need exists for a nano formulation containing two active molecules for the treatment of glaucoma that can be delivered topically as eye drops thereby circumventing the side effects associated with conventional eye drop formulations and providing higher drug concentration. Also, a need exists for an elastic nano-vesicles for co-delivery of antiglaucoma agents and siRNA for treatment of glaucoma and method of preparing the same, as described in greater detail herein.
OBJECTS OF THE INVENTION
The foremost object of the invention is to enhance the corneal permeability and consequently the antiglaucoma efficacy of active agent(s).
It is yet another object of the present invention to develop a formulation to allow for controlled delivery of active agents(s).
It is yet another object of the present invention to develop a formulation to allow for controlled and targeted delivery of active agent(s).
It is yet another object of the present invention to overcome the limitations of conventional eye drop formulations of active agent(s).
It is an object of the present invention to deliver active agent(s) using dendrimeric peptide which can break down into components of nil to low toxicity.
It is an object of the present invention to enhance the targeting efficiency of the formulation using suitable targeting ligand.
It is still an object of the present invention to provide formulations using components that are physically and chemically compatible.
It is yet an object of the present invention to deliver active agent(s) for producing requisite pharmacological action.
It is further an object of the present invention to deliver the pharmaceutical formulation in the form of carriers, which could augment the efficacy and safety of the encapsulated active agent(s).
These and other objects and features of the present invention will become readily apparent to one skilled in the art from the detailed description given hereafter.
SUMMARY OF THE INVENTION
The following summary is provided to facilitate an understanding of some of the innovative features unique to the disclosed embodiment and is not intended to be a full description.
It is, therefore, one aspect of the disclosed embodiments to provide for an improved nano formulation containing two active molecules for the treatment of glaucoma that can be delivered topically as eye drops thereby circumventing the side effects associated with conventional eye drop formulations and providing higher drug concentration.
It is another aspect of the disclosed embodiments to provide for an improved method for use of dendrimeric peptide attached to nano-vesicles (NV) loaded with active agent(s) for enhancing the corneal permeability of the formulation and ii) active agent(s) loaded nano-vesicles coated with targeting ligand (HA) for improving the targeting efficiency into the posterior segments of the eye.
It is further aspect of the disclosed embodiments to provide for an improved elastic nano-vesicles for co-delivery of antiglaucoma agents and siRNA for treatment of glaucoma and method of preparing the same.
Elastic nano-vesicles for co-delivery of antiglaucoma agents and siRNA for treatment of glaucoma and method of preparing thereof. The novel formulation containing the mixture of two types of nano-vesicles viz., first nano-vesicles containing of an active agent (TM) and conjugated with DP, second nanovesicles containing an active agent (siRNA) and coated with HA. The dendrimeric peptide (DP) consists of branched macromolecule(s) containing peptide core and/ or peripheral peptide chains.
As used herein, the term ‘active agent’ refers to an agent, including but not limited to small molecule drug, protein, peptide, siRNA, mRNA, nucleic acid (nucleotides, nucleosides, and analogues thereof), prodrug, which may provide prophylactic action, nutritive effect, pharmacological or therapeutic action or treatment of disease upon administration to a subject (human or non-human animal) either alone or in combination with other active or inactive components.
The carrier may be selected from the class of natural, semi-synthetic or synthetic phospholipids, polymers, lipids, carbohydrates, polysaccharides, ionic or non-ionic surfactants, peptides and/ or their derivatives.
The preparation may also contain other formulation components for the purpose of preparation. The ligand may refer to an agent, including but not limited to small molecule drug, proteins, peptide, aptamer, nucleic acid (nucleotides, nucleosides, and analogues thereof), and similar agents which are over expressed in glaucoma and facilitate active targeting of nanoparticulate carriers to retinal ganglion cells.
In one embodiment, the DP has sequence Gly-Leu-Lys-(Lys-(Arg)2)2 (C-N terminus). In preferred compositions, the DPs or ligands are conjugated on the surface of nano-vesicles loaded with TM or siRNA. In especially preferred compositions, the carriers are nanoparticulate in nature with size < 1000 nm comprised matrix of phospholipids or surfactant or polymer or any other agent. In particularly preferred compositions, the aforementioned nano-vesicles additionally comprise edge activator(s) which provide elasticity to the carrier. In particularly preferred compositions, the abovementioned nano-vesicles comprised of non-ionic surfactant(s). The nanoparticulate system contains two types of elastic nano-vesicles loaded with two actives (TM and siRNA) separately.
For the fabrication of first type of elastic nano-vesicular system - DP was synthesized by solid phase peptide synthesis and purified by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC). The synthesized DP was characterized by Mass Spectrometry (MS), Nuclear magnetic resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DSC). The Elastic nano-vesicles of active agent(s) were prepared using ethanol injection method containing surfactant (Span 60) and edge activator (Tween 80). The DP was conjugated on the vesicle surface by EDC-NHS conjugation. Solid-state characterization, involving physical and chemical compatibility studies between TM and components of the formulation were performed by Fourier Transform Infrared (FTIR) Spectroscopy and Differential Scanning Calorimetry (DSC).
The siRNA was selected based on the suggestions by maximum siRNA designing software (siPRED, siDirect, Block-iT, GeneScript and Eurofins Genomics). The siRNAs that target the various regions of caspase 2 gene mRNA were designed based on the percent inhibition efficiency of 82.1%, Guanine-Cytosine (GC) content of 47% and no self-complementarity (absence of potential hairpin formation and self-annealing sites). The sequence of the sense strand of designed siRNA was 5'ACCUCCUAGAGAAGGACAU[dT][dT]3' and antisense strand was 5'AUGUCCUUCUCUAGGAGGU [dT][dT]3'.
The designed siRNA sequence was blasted against the mouse genome database to eliminate the cross-silence phenomenon with non-target genes. Scrambled siRNA that does not target any gene was used as the negative control siRNA. The optimum annealing temperature for all the primers was confirmed at 55 °C.
For the fabrication of second type of elastic nano-vesicle system - The elastic nanocarrier dispersion without TM was prepared and stabilized by polycationic polymer Polyethyleneimine (PEI). The unconjugated PEI was removed by centrifugation and the resultant dispersion was lyophilized using a cryoprotectant. The particle size (PS), zeta potential (ZP) and polydispersity index (PDI) of the resultant mixture were determined using Malvern Zeta Sizer. The lyophilized PEI-coated nanocarrier/ nano-vesicle was resuspended in molecular biology grade water and the resultant dispersion was added dropwise into a stirred solution of siRNA and cationized HA under aseptic condition using Laminar air flow cabinet. The unbound polyelectrolyte was removed by centrifugation and lyophilized using a cryoprotectant. The PS, ZP, and PDI of the final lyophilized siRNA conjugated nano-vesicle were determined using Malvern Zeta Sizer. The molar siRNA concentration was determined by using Epoch 2 microplate reader.
Characterization of the elastic nano-vesicle formulations viz. TM nano-vesicles without DP/ ligand (HA), TM nano-vesicles with DP and without HA, siRNA nano-vesicles without DP/ HA and siRNA nano-vesicles with HA and without DP was done with respect to particle size, PDI, ZP and entrapment efficiency (EE%). Surface morphology of the nano-vesicles was studied by Transmission electron microscopy (TEM). X-ray diffraction studies were performed to determine the amorphous nature of the nano-vesicles. Drug release study was performed in phosphate buffer saline using dialysis method. In vitro cytotoxicity assessment and cell uptake studies were performed on HCE-2 (human corneal epithelial cells) and RGC-5 cells (retinal ganglion cells). Caspase 2 gene expression studies were assessed by quantitative real-time PCR (qRT-PCR). Eye irritation study, histopathology study and anti-glaucoma efficacy studies were performed in male Sprague Dawley (SD) rats.
The DP was found to exhibit – i) molecular weight of 1197.5 g/mol evident from the MS investigation, ii) single peak purity from the RP-HPLC chromatogram and iii) designated structure from the NMR spectrum. FT-IR and DSC studies revealed absence of incompatibility between TM and excipients of the formulation. The particle size, PDI, ZP and EE% of TM nano-vesicles without DP/ HA was found to be 178.9±4.21 nm, 0.202±0.018, -35.5±0.44 mV and 58.68%±3.12 respectively. The particle size, PDI, ZP and EE% of TM nano-vesicles with DP and without HA was found to be 282.3±3.96 nm, 0.305±0.002, 22.6±0.87 mV and 53.21%±4.86 respectively. The particle size, PDI, ZP and EE% of siRNA nano-vesicles without DP/ HA was found to be 303.4±2.721 nm, 0.438±0.014, 32.5±0.331 mV and 56.11%±4.24 respectively. The particle size, PDI, ZP and EE% of siRNA nano-vesicles with HA and without DP was found to be 495.6±5.15 nm, 0.411±0.316, -41.3±0.81 mV and 51.57%±5.03 respectively.
The XRD spectrum of TM showed characteristic peaks at 2?=21.46° and most intense peak at 14.26° indicating the crystalline nature of the drug. Contrarily, the XRD spectra of optimized nano-vesicles showed a crystalline peak at 21.44°; however, the intense peak of TM was missing. This could be indicative of a partial amorphization of the entrapped drug within the nano-vesicles. Further, DP-conjugated nano-vesicles showed broad halo region between 20° and 25° which indicates the amorphous nature of the nano-vesicles.
Drug release studies of free TM and nano-vesicles formulations were carried out in pH 7.4 phosphate buffer by dialysis method. Free TM demonstrated an early burst release whereas the nano-vesicle formulations exhibited a sustained release over 24 h. The cumulative percentage of drug released after 24 h for nano-vesicles was observed to be 57.45%, whereas 76.55% of TM was released from free TM solution after 24 h. The sustained drug release in nano-vesicles may be due to the presence of an alkyl chain in Tween 80 which increases the hydrophobicity of the vesicles and the high transition temperature of Span 60 that forms a less permeable and more rigid bilayer.
The results of ex vivo permeation studies showed that the amount of TM permeated from elastic nano-vesicles through the bovine cornea was higher compared to the free TM and marketed formulation. This may be due to the presence of Tween 80 which was used as an edge activator in the formulation of nano-vesicles. The bilayer structure upon hydration of the vesicles would become loose and more flexible due to the high aqueous solubility of Tweens, leading to increased permeability to solutes. The DP conjugated nano-vesicles (DP-NVs-TM) showed enhanced permeation through the bovine cornea as compared to unconjugated/ plain vesicles (NVs-TM). This could be due to the strong cationic nature of the amine-terminated dendrimeric peptide which possibly interacts with the negatively charged corneal membrane and results in enhanced permeation of the vesicles.
In vitro cytotoxicity study suggested that against HCE-2 cell lines, the test compounds (free TM solution, TM nano-vesicles and DP conjugated nano-vesicles) showed non-toxic potential properties with the % cell viability of 98.03%, 91.46% and 79.81% at the highest concentration of 3 mg/ml respectively. In the case of RGC-5 cells, the statistical data observations from the cytotoxicity study suggested that the test compounds viz., HA-coated nano-vesicles and HA-coated siRNA loaded nano-vesicles showed non-toxic potential properties with the % cell viability of 96.16% and 83.01% at the highest concentration of 3 mg/ml respectively.
The cell uptake of free FITC dye, FITC labelled plain nano-vesicles, FITC labelled DP conjugated nano-vesicles and FITC labelled HA coated nano-vesicles was studied using flow cytometry in RGC-5 cells after 24 h of incubation. The RGC-5 cells showed high fluorescence intensity in the formulated nano-vesicles compared to free FITC dye. The mechanism behind the internalization of nano-vesicles in RGC-5 is the squeezing of the elastic vesicles across the cornea and interaction of hydrophilic edge activators with the aqueous and vitreous humour as they are majorly constituted of water (~90%). It was observed that the DP conjugation to the nano-vesicles resulted in higher cellular uptake by RGC-5 cells in comparison to the plain nano-vesicles. The reason for the enhanced uptake of DP-conjugated nano-vesicles may be due to the ability of the dendrimeric peptides to enhance the uptake of the drug via the enterocytes. The dendrimers are also reported to formation of holes in the lipid bilayer and loosen the epithelial cell junctions which change the physical properties of the cell membrane leading to the solubilization of the corneal membrane. The presence of amino groups in DP such as positively charged arginine residues enhances the uptake of nano-vesicles via the electrostatic interactions with the negatively charged phospholipid layer. Similarly, it was observed that coating the nano-vesicles with HA resulted in higher cellular uptake by RGC-5 cells in comparison to the uncoated one. CD44 is a widely expressed transmembrane glycoprotein present throughout the retina (retinal pigment epithelial cells, muller cells and retinal ganglion cells) and cell surface receptor for HA. HA increases the drug’s residence duration on the ocular surface, reduces the loss of the drug and increases the bioavailability of the drug due to the interaction with the precorneal mucin layer by non-covalent bonding.
The results of the caspase 2 gene expression studies suggest that the relative mRNA expression values (relative fold change) of the caspase-2 gene were down-regulated in the formulations comprising of optimized positive siRNA (0.025±0.072) as well as naked positive siRNA (0.180±0.098) but the optimized negative siRNA (1.250±0.102) treatment group was up-regulated compared to the untreated group (1.000±0.310). The results confirmed that the HA coated siRNA loaded nano-vesicles effectively inhibited Caspase-2 gene expression with relative fold change value of 0.025±0.07 in transfected RGC-5 cells as compared to other treatment groups.
The results of the eye irritation study revealed that no premature cell death or cell degeneration related to the treatment indicating the formulations to be safe and non-irritant. Histopathological examination of the iris, ciliary body and retina did not show any histologic changes like atrophy, inflammation and vacuolation. The cell layers in all the tissues were organized and there was no change in tissue architecture. Together with all the above examinations, it proved that all the tested formulations had good corneal biocompatibility and the formulation was non-irritant. Pharmacodynamic studies in male Sprague Dawley rats revealed that treatment with DP conjugated TM loaded elastic nano-vesicles exerted a stronger effect on IOP reduction (IOP reduced from 26 mm Hg to 6 mm Hg) compared to non-conjugated TM loaded nano-vesicles (IOP reduced from 23 mm Hg to 9 mm Hg) and commercial formulation (IOP reduced from 25 mm Hg to 10 mm Hg). These findings form the first report on the applicability of dendrimeric peptide to bring about enhanced uptake of active agent(s).
DETAILED DESCRIPTION
The values and configurations discussed in these non-limiting examples can be varied and are cited merely to illustrate at least one embodiment and are not intended to limit the scope thereof.
The embodiments now will be described more fully hereinafter with reference to the accompanying drawings, in which illustrative embodiments of the invention are shown. The embodiments disclosed herein can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Like numbers refer to like elements throughout. As used herein, the term "and/or" includes all combinations of one or more of the associated listed items.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
Invention: Elastic nano-vesicles for co-delivery of antiglaucoma agents and siRNA for treatment of glaucoma and method of preparing thereof. The novel formulation containing the mixture of two types of nano-vesicles viz., first nano-vesicles containing of an active agent (TM) and DP, second nanovesicles containing of an active agent (siRNA) and coated with HA . The dendrimeric peptide (DP) consists of branched macromolecule(s) containing peptide core and/ or peripheral peptide chains.
As used herein, the term ‘active agent’ refers to an agent, including but not limited to small molecule drug, protein, peptide, siRNA, mRNA, nucleic acid (nucleotides, nucleosides, and analogues thereof), prodrug, which may provide prophylactic action, nutritive effect, pharmacological or therapeutic action or treatment of disease upon administration to a subject (human or non-human animal) either alone or in combination with other active or inactive components.
The carrier may be selected from the class of natural, semi-synthetic or synthetic phospholipids, polymers, lipids, carbohydrates, polysaccharides, ionic or non-ionic surfactants, peptides and/ or their derivatives.
The preparation may also contain other formulation components for the purpose of preparation. The ligand may refer to an agent, including but not limited to small molecule drug, proteins, peptide, aptamer, nucleic acid (nucleotides, nucleosides, and analogues thereof), and similar agents which are over expressed in glaucoma and facilitate active targeting of nanoparticulate carriers to retinal ganglion cells.
In one embodiment, the DP has sequence Gly-Leu-Lys-(Lys-(Arg)2)2 (C-N terminus). FIG. 1 illustrates the structure of DP depicting the sequence Gly-Leu-Lys-(Lys-(Arg)2)2. In preferred compositions, the DPs or ligands are conjugated on the surface of nano-vesicles loaded with TM or siRNA. In especially preferred compositions, the carriers are nanoparticulate in nature with size < 1000 nm comprised matrix of phospholipids or surfactant or polymer or any other agent. In particularly preferred compositions, the aforementioned nano-vesicles additionally comprise edge activator(s) which provide elasticity to the carrier. In particularly preferred compositions, the abovementioned nano-vesicles comprised of non-ionic surfactant(s). The nanoparticulate system contains two types of elastic nano-vesicles loaded with two actives (TM and siRNA) separately.
For the fabrication of first type of elastic nano-vesicular system - DP was synthesized by solid phase peptide synthesis and purified by Reverse Phase-High Performance Liquid Chromatography (RP-HPLC). The synthesized DP was characterized by Mass Spectrometry (MS), Nuclear magnetic resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DSC). The Elastic nano-vesicles of active agent(s) were prepared using ethanol injection method containing surfactant (Span 60) and edge activator (Tween 80). The DP was conjugated on the vesicle surface by EDC-NHS conjugation. Solid-state characterization, involving physical and chemical compatibility studies between TM and components of the formulation were performed by Fourier Transform Infrared (FTIR) Spectroscopy and Differential Scanning Calorimetry (DSC).
FIG. 3 illustrates the Differential Scanning Calorimetric thermograms of (A) DP, (B) Plain TM loaded nano-vesicles and (C) DP conjugated TM loaded nano-vesicles. The DSC thermogram of DP (FIG. 3A) showed endothermic peaks in the range of 200-230 °C corresponding to the melting points of amino acids i.e., glycine at 233°C, lysine 215°C and arginine at 222 °C. In the DSC thermogram of TM loaded nano-vesicles (FIG. 3B) the drug endothermic peak was not visible but Span 60 and mannitol melting point peaks were found at 51 °C and 158 °C respectively. The absence of TM endotherm in formulation indicated the loss of crystallinity of the drug, which might have molecularly dispersed in the surfactant (Span 60) matrix. The further broad and the less intense peak were observed in the case of DP conjugated nano-vesicles (FIG. 3C) at 115 °C which could be due to amine terminated dendrimer.
FIG. 4 illustrates FTIR spectra of (A) DP, (B) TM loaded nano-vesicles, (C) DSPE-PEG-COOH functionalized nano-vesicles and (D) DP conjugated nano-vesicles. The FTIR spectrum of DP (Fig. 4A) showed an intense sharp peak at 1659.53 cm-1 of the (-C=O) carbonyl group of DP. The peak at 1538.50 cm-1 was observed due to the bending vibration of the hydroxyl group of arginine. The broad peak due to the aliphatic group (-CH2) of lysine was observed at a wavenumber of 3275.96 cm-1. The FTIR spectrum of TM loaded nano-vesicles (Fig. 4B) showed the peaks of TM as well as the excipients. In the FTIR spectrum of DSPE-PEG-COOH functionalized nano-vesicles (Fig. 4C) showed a sharp -C=O peak of ester at 1735 cm-1. The contribution of two ester groups from Span 60 and two ester groups from DSPE-PEG-COOH resulted in a null neighboring effect due to similar neighboring ester groups and the peak sharpness. The two small peaks corresponding to acid (-C=O) were observed at 1637 cm-1 and amide (-C=O) was observed at 1584 cm-1. In the FTIR spectrum of DP conjugated nano-vesicles (Fig. 4D), alkane -CH groups were observed at 2849 cm-1 and 2916 cm-1. A sharp ester -C=O peak, the same as in unconjugated nano-vesicles was observed at 1735 cm-1 but less intense than the peak in unconjugated nano-vesicles. The formation of amide bond -CONH was confirmed with the peak at 1674 cm-1 and its shape was distorted due to multiple -CONH groups from peptide dendrimer and the result of conjugation between linker and dendrimeric peptide. The broad peak of -NH and -OH groups were observed at 3355 cm-1 and 3251 cm-1.
FIG. 5 illustrates the transmission electron photomicrographs of nano-vesicles. Fig. 5A) Plain nano-vesicles (NVs), Fig. 5B) DP conjugated nano-vesicles (DP-NVs). The particle size was in accordance with the results obtained with dynamic light scattering (DLS) method. The size of NVs was observed to be >150 nm, however the size of DP conjugated nano-vesicles was observed to be > 200 nm which could be attributed due to conjugation of DP.
The siRNA was selected based on the suggestions by maximum siRNA designing software (siPRED, siDirect, Block-iT, GeneScript and Eurofins Genomics). The siRNAs that target the various regions of caspase 2 gene mRNA were designed based on the percent inhibition efficiency of 82.1%, Guanine-Cytosine (GC) content of 47% and no self-complementarity (absence of potential hairpin formation and self-annealing sites). The sequence of the sense strand of designed siRNA was 5' ACCUCCUAGAGAAGGACAU[dT][dT]3' and antisense strand was 5'AUGUCCUUCUCUAGGAGGU [dT][dT]3'.
The designed siRNA sequence was blasted against the mouse genome database to eliminate the cross-silence phenomenon with non-target genes. Scrambled siRNA that does not target any gene was used as the negative control siRNA. The optimum annealing temperature for all the primers was confirmed at 55 °C.
For the fabrication of second type of elastic nano-vesicle system - The elastic nanocarrier dispersion without TM was prepared and stabilized by polycationic polymer Polyethyleneimine (PEI). The unconjugated PEI was removed by centrifugation and the resultant dispersion was lyophilized using a cryoprotectant. The PS, ZP and PDI of the resultant mixture were determined using Malvern Zeta Sizer. The lyophilized PEI-coated nanocarrier/ nano-vesicle was resuspended in molecular biology grade water and the resultant dispersion was added dropwise into a stirred solution of siRNA and cationized HA under aseptic condition using Laminar air flow cabinet. The unbound polyelectrolyte was removed by centrifugation and lyophilized using a cryoprotectant. The PS, ZP, and PDI of the final lyophilized siRNA conjugated nano-vesicle were determined using Malvern Zeta Sizer. The molar siRNA concentration was determined by using Epoch 2 microplate reader.
Working of the invention: Characterization of the elastic nano-vesicle formulations viz. TM nano-vesicles without DP/ ligand (HA), TM nano-vesicles with DP and without HA, siRNA nano-vesicles without DP/ HA and siRNA nano-vesicles with HA and without DP was done with respect to particle size, polydispersity index (PDI), zeta potential (ZP) and entrapment efficiency (EE%). Surface morphology of the nano-vesicles was studied by Transmission electron microscopy (TEM). X-ray diffraction studies were performed to determine the amorphous nature of the nano-vesicles. Drug release study was performed in phosphate buffer saline using dialysis method. In vitro cytotoxicity assessment and cell uptake studies were performed on HCE-2 (human corneal epithelial cells) and RGC-5 cells (retinal ganglion cells). Caspase 2 gene expression studies were assessed by quantitative real-time PCR (qRT-PCR). Eye irritation study, histopathology study and anti-glaucoma efficacy studies were performed in male Sprague Dawley (SD) rats.
The DP was found to exhibit – i) molecular weight of 1197.5 g/mol evident from the MS investigation, ii) single peak purity from the RP-HPLC chromatogram and iii) designated structure from the NMR spectrum. FT-IR and DSC studies revealed absence of incompatibility between TM and excipients of the formulation.
FIG. 2 illustrates the H NMR spectrum of (A) DP with the chemical shift assignments for the different groups (ppm), (B) Plain TM loaded nano-vesicles and (C) DP conjugated TM loaded nano-vesicles. The H1NMR spectra of DP (FIG. 2A) showed a doublet peak at a signal of 0.8 ppm representing the hydrogens of the isopropyl group of leucine. Chemical shifts were observed at 2.5 ppm and between 3.1 to 3.6 ppm due to the protons of the methylene (CH2-NH) and methine groups (-CH2-NH-CH=NH) of arginine. The chemical shift was observed between 4.21 to 4.32 ppm due to the protons of the methine groups attached next to NH and -NH2 groups. The NMR spectrum of the DP conjugated nano-vesicles (FIG. 2C) showed additional peaks between 4 ppm to 4.5 ppm due to the increased protons attached to the amino acids of the peptide dendrimer as compared to the NMR spectrum of the plain nano-vesicles (FIG. 2B).
The particle size, PDI, ZP and EE% of TM nano-vesicles without DP/ HA was found to be 178.9±4.21 nm, 0.202±0.018, -35.5±0.44 mV and 58.68%±3.12 respectively. The particle size, PDI, ZP and EE% of TM nano-vesicles with DP and without HA was found to be 282.3±3.96 nm, 0.305±0.002, 22.6±0.87 mV and 53.21%±4.86 respectively. The particle size, PDI, ZP and EE% of siRNA nano-vesicles without DP/ HA was found to be 303.4±2.721 nm, 0.438±0.014, 32.5±0.331 mV and 56.11%±4.24 respectively. The particle size, PDI, ZP and EE% of siRNA nano-vesicles with HA and without DP was found to be 495.6±5.15 nm, 0.411±0.316, -41.3±0.81 mV and 51.57%±5.03 respectively.
FIG. 6 illustrates XRD patterns of A) Free TM, B) TM nano-vesicles (NVs), C) DP conjugated nano-vesicles (DP-NVs). Characteristic and intense peaks were observed in the XRD spectrum of TM at 2?=21.46° and 14.26° indicating the crystalline nature of the drug. The NVs showed less intense peak at 21.2°, indicating the partial amorphization of the entrapped drug within the nano-vesicles. Further, DP conjugated nano-vesicles showed broad, halo region between 20° and 25° which indicates the amorphous nature of the nano-vesicles.
The XRD spectrum of TM showed characteristic peaks at 2?=21.46° and most intense peak at 14.26° indicating the crystalline nature of the drug. Contrarily, the XRD spectra of optimized nano-vesicles showed a crystalline peak at 21.44°; however, the intense peak of TM was missing. This could be indicative of a partial amorphization of the entrapped drug within the nano-vesicles. Further, DP-conjugated nano-vesicles showed broad halo region between 20° and 25° which indicates the amorphous nature of the nano-vesicles.
Drug release studies of free TM and nano-vesicles formulations were carried out in pH 7.4 phosphate buffer by dialysis method. Free TM demonstrated an early burst release whereas the nano-vesicle formulations exhibited a sustained release over 24 h. The cumulative percentage of drug released after 24 h for nano-vesicles was observed to be 57.45%, whereas 76.55% of TM was released from free TM solution after 24 h. The sustained drug release in nano-vesicles may be due to the presence of an alkyl chain in Tween 80 which increases the hydrophobicity of the vesicles and the high transition temperature of Span 60 that forms a less permeable and more rigid bilayer.
The results of ex vivo permeation studies showed that the amount of TM permeated from elastic nano-vesicles through the bovine cornea was higher compared to the free TM and marketed formulation. This may be due to the presence of Tween 80 which was used as an edge activator in the formulation of nano-vesicles. The bilayer structure upon hydration of the vesicles would become loose and more flexible due to the high aqueous solubility of Tweens, leading to increased permeability to solutes. The DP conjugated nano-vesicles (DP-NVs-TM) showed enhanced permeation through the bovine cornea as compared to unconjugated/ plain vesicles (NVs-TM). This could be due to the strong cationic nature of the amine-terminated dendrimeric peptide which possibly interacts with the negatively charged corneal membrane and results in enhanced permeation of the vesicles.
In vitro cytotoxicity study suggested that against HCE-2 cell lines, the test compounds (free TM solution, TM nano-vesicles and DP conjugated nano-vesicles) showed non-toxic potential properties with the % cell viability of 98.03%, 91.46% and 79.81% at the highest concentration of 3 mg/ml respectively. In the case of RGC-5 cells, the statistical data observations from the cytotoxicity study suggested that the test compounds viz., Hyaluronic acid-coated nano-vesicles and HA-coated siRNA loaded nano-vesicles showed non-toxic potential properties with the % cell viability of 96.16% and 83.01% at the highest concentration of 3 mg/ml respectively.
The cell uptake of free FITC dye, FITC labelled plain nano-vesicles, FITC labelled DP conjugated nano-vesicles and FITC labelled HA coated nano-vesicles was studied using flow cytometry in RGC-5 cells after 24 h of incubation. The RGC-5 cells showed high fluorescence intensity in the formulated nano-vesicles compared to free FITC dye. The mechanism behind the internalization of nano-vesicles in RGC-5 is the squeezing of the elastic vesicles across the cornea and interaction of hydrophilic edge activators with the aqueous and vitreous humour as they are majorly constituted of water (~90%). It was observed that the DP conjugation to the nano-vesicles resulted in higher cellular uptake by RGC-5 cells in comparison to the plain nano-vesicles. The reason for the enhanced uptake of DP-conjugated nano-vesicles may be due to the ability of the dendrimeric peptides to enhance the uptake of the drug via the enterocytes. The dendrimers are also reported to formation of holes in the lipid bilayer and loosen the epithelial cell junctions which change the physical properties of the cell membrane leading to the solubilization of the corneal membrane. The presence of amino groups in DP such as positively charged arginine residues enhances the uptake of nano-vesicles via the electrostatic interactions with the negatively charged phospholipid layer. Similarly, it was observed that coating the nan-vesicles with HA resulted in higher cellular uptake by RGC-5 cells in comparison to the uncoated one. CD44 is a widely expressed transmembrane glycoprotein present throughout the retina (retinal pigment epithelial cells, muller cells and retinal ganglion cells) and cell surface receptor for HA. HA increases the drug’s residence duration on the ocular surface, reduces the loss of the drug and increases the bioavailability of the drug due to the interaction with the precorneal mucin layer by non-covalent bonding.
The results of the caspase 2 gene expression studies suggest that the relative mRNA expression values (relative fold change) of the caspase-2 gene were down-regulated in the formulations comprising of optimized positive siRNA (0.025±0.072) as well as naked positive siRNA (0.180±0.098) but the optimized negative siRNA (1.250±0.102) treatment group was up-regulated compared to the untreated group (1.000±0.310). The results confirmed that the HA coated siRNA loaded nano-vesicles effectively inhibited Caspase-2 gene expression with relative fold change value of 0.025±0.07 in transfected RGC-5 cells as compared to other treatment groups.
The results of the eye irritation study revealed that no premature cell death or cell degeneration related to the treatment indicating the formulations to be safe and non-irritant. Histopathological examination of the iris, ciliary body and retina did not show any histologic changes like atrophy, inflammation and vacuolation. The cell layers in all the tissues were organized and there was no change in tissue architecture. Together with all the above examinations, it proved that all the tested formulations had good corneal biocompatibility and the formulation was non-irritant. Pharmacodynamic studies in male Sprague Dawley rats revealed that treatment with DP conjugated TM loaded elastic nano-vesicles exerted a stronger effect on IOP reduction (IOP reduced from 26 mm Hg to 6 mm Hg) compared to non-conjugated TM loaded nano-vesicles (IOP reduced from 23 mm Hg to 9 mm Hg) and commercial formulation (IOP reduced from 25 mm Hg to 10 mm Hg). These findings form the first report on the applicability of dendrimeric peptide to bring about enhanced uptake of active agent(s).
Other working results: FIG. 7 illustrates In vitro release studies of free TM solution (Free TM) and TM-loaded nano-vesicles (NVs-TM) in phosphate buffered saline pH 7.4. The values are expressed as mean ± SD, n=3. Free TM demonstrated an early burst release whereas the nano-vesicle dispersion showed the sustained release of the drug for a period of 24 h. The cumulative percentage of drug released after 24 h for nano-vesicles was observed to be 57.45%, whereas 76.55% of TM was released from free TM solution after 24 h.
FIG. 8 illustrates the comparison of cumulative % of drug permeated from formulations in Ex vivo corneal permeation studies using bovine cornea. The results showed that the amount of TM permeated from nano-vesicles through the bovine cornea was higher compared to the free TM and marketed formulation. This may be due to the presence of Tween 80 which was used as an edge activator in the formulation of nano-vesicles. The DP conjugated nano-vesicles (DP-NVs-TM) showed enhanced permeation through the bovine cornea as compared to unconjugated nano-vesicles (NVs-TM). This could be due to the strong cationic nature of the amine-terminated dendrimeric peptide which possibly interacts with the negatively charged corneal membrane and results in enhanced permeation of the nano-vesicles.
FIG. 9 illustrates the quantitative flow cytometric analysis of cellular uptake of untreated, plain FITC, FITC labelled elastic nano-vesicles without Hyaluronic acid (HA), HA-coated nano-vesicles, DP conjugated TM loaded nano-vesicles in RGC-5 cells after 24 h incubation. The RGC-5 cells showed high fluorescence intensity in the formulated nano-vesicles (NV+TM) compared to free FITC dye. The mean fluorescence uptake of conjugated nano-vesicles (NV+TM+DP) was significantly higher than the plain nano-vesicles (NV+TM) implying enhanced uptake of TM within the cells by virtue of their conjugation with DP. Similarly, it was observed that coating the nano-vesicles with HA resulted in higher cellular uptake by RGC-5 cells in comparison to the uncoated one (NV+TM).
FIG. 10 illustrates the relative mRNA expression of Caspase-2 genes in transfected Mouse retinal ganglion (RGC-5) cells in different culture groups by RTPCR. The obtained results suggest that the relative gene expression level of the Caspase-2 gene was down-regulated in the Optimized positive siRNA as well as naked positive siRNA but the Optimized Negative siRNA treatment group was up-regulated compared to the untreated group.
FIG. 11 illustrates the acute eye irritation microscopic images of rat eyes treated with 0.1 mL of 0.5% w/v Timolol maleate solution (Group I), optimized nano-vesicles formulation (Group II) and DP-conjugated nano-vesicles formulation (Group III). Sodium fluorescein was used for the initial detection of breaks in the corneal epithelium after 1 h, 24 h and 48 h of treatment. After 72 h of treatment, the eyes were observed for any cell degeneration or cell death by staining with Lissamine green.
FIG. 12 illustrates the chronic eye irritation microscopic images of rat eyes treated 5 times with 0.1 mL for 1 week with 0.5% w/v Timolol maleate solution (Group I), optimized nano-vesicles formulation (Group II) and DP-conjugated nano-vesicles formulation (Group III). Sodium fluorescein was used for the initial detection of breaks in the corneal epithelium after 1 h, 24 h and 48 h of treatment. After 72 h of treatment, the eyes were observed for any cell degeneration or cell death by staining with Lissamine green.
FIG. 13 illustrates the histological cross-sections stained with Haematoxylin and Eosin of SD rat’s cornea, iris, ciliary body and retina of negative control (Group I), 0.5% w/v TM solution (Group II), optimized nano-vesicles formulation (Group III) and DP-conjugated nano-vesicles formulation (Group IV) for 1 week (original magnification, 100x)
FIG. 14 illustrates the IOP lowering effect after administration of normal saline (Group II: Positive control), marketed formulation (Group III: MF), TM loaded nano-vesicles formulation (Group IV: NVs) and DP conjugated nano-vesicles formulation (Group V: DP-NVs). Group I represent the negative control i.e., untreated group.
Best method of use of the invention with examples: The present invention relates to elastic nano-vesicles with/ without dendrimeric peptide, ligand (HA), siRNA and drug(s). The nano- vesicles and other agents, which help in making the complete nano-system, could be administered either along with the active agent(s) in a carrier or associated with the active agent(s) in a pharmaceutically acceptable base. Additionally, the carrier could be integrated in a pharmaceutically acceptable base. Following is the comprehensive procedure for the formulation of the present invention and can be easily understood by anyone skilled in the art.
Example 1: In this example, the method of synthesis of the DPs is explained. The arginine (Arg)- terminated dendrimeric peptide was synthesized by Fluorenyl methoxy carbonyl protecting group (Fmoc) solid phase peptide synthesis (SPSS) from C to N-terminal of Glycine-Leucine-Lysine-(Lysine-(Arginine)2)2. For the synthesis of DP, the chlorotrityl chloride resin was activated using piperidine (20% v/v) in dimethylformamide (DMF) which was used to swell the resin. Post activation the resin was washed with DMF. Further, HBTU and DIEA-activated Fmoc-Gly-OH were coupled with the resin and treated with piperidine (20% v/v). The final product was washed with DMF before the coupling of the subsequent amino acid was carried out. The next amino acid was coupled to the previous amino acids after achieving good coupling efficiency. The above method was continuously repeated until the desired generation of dendrimer was obtained. Finally, to cleave off the formed DP from the resin it was treated with piperidine in DMF. The dried resin was kept for stirring and the residue obtained was azeotroped with toluene and triturated with diethyl ether. Finally, the obtained residue was dissolved in deionized water and the resultant DP was stored at 2-8 °C until further use. The DPs were then purified using preparative RP-HPLC and characterized by MS, NMR and DSC analysis.
Example 2: In this example, the method of preparation of the nano-vesicles containing DP with active agent(s) is described. The DP was conjugated onto elastic nano-vesicles containing TM. The nano-vesicles were developed using the ethanol injection method, followed by size reduction using sonication. The materials comprised: surfactant (2-[(2R,3S,4R)-3,4-dihydroxyoxolan-2-yl]-2-hydroxyethyl octadecenoate phospholipid (SPAN 60; 70-90 mg), edge activator (Polyoxyethylene (80) sorbitan monooleate (TWEEN 80); 10-30 mg), organic phase (ethanol) and aqueous phase (water) (1:4) and the drug TM (100 mg). The surfactants and drug were dissolved in ethanol and then subjected to sonication for 1 min over a water bath. The resultant alcoholic solution was then injected into a preheated (70 °C) aqueous phase containing an edge activator at a rate of 1 mL/min. The resultant dispersion was stirred for 30 min at 70 °C to remove traces of ethanol and 30 min at room temperature. Finally, the dispersion was sonicated in a water bath for 4 min to avoid vesicle aggregation. Free TM was removed by centrifuging the nano-vesicle dispersion at 20,000 rpm for 30 min at 4 °C.
Surface conjugation of drug loaded nano-vesicles was carried out by covalent conjugation using preformed nano-vesicles. The DP was covalently bound to the carboxyl functionalized nano-vesicles (NVs-DSPE-PEG-COOH2000) using EDC-NHS chemistry (1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), N-Hydroxysuccinimide (NHS). The carboxyl functionalized nano-vesicles were prepared by replacing 5% of Span 60 with that of DSPE-PEG-COOH2000. To NVs-DSPE-PEG-COOH2000 dispersion, EDC was added dropwise and kept for stirring for 45 min at room temperature. To this reaction mixture, NHS (EDC: NHS in the ratio of 2:1) was added and stirred at room temperature for 12 h. The unreacted EDC-NHS was removed by dialysis and 5 times more of DSPE-PEG-COOH2000 of dendrimeric peptide was added and stirred for 24 h for the accomplishment of conjugation. The excess amount of dendrimeric peptide was removed by dialysis and the obtained conjugated dispersion of nano-vesicles was lyophilized using 2.5% cryoprotectant (mannitol).
Example 3: In this example, the method of preparation of the nanocarrier/ nano-vesicle containing targeting ligand (HA) with active agent(s) is described. One of the active agents, siRNA, targeting the various regions of caspase 2 gene was designed using siPRED, siDirect, siRNA Target Finder, Block-iT RNAi Designer and Eurofins Genomics. The siRNA was designed based on the percent inhibition efficiency (>80%), the desirable GC content (30-52% according to Reynold et al rule (Reynolds et al., 2004), and the absence of self-complementarity. The sequence of the sense strand of designed siRNA was 5' ACCUCCUAGAGAAGGACAU[dT][dT]3' and antisense strand was 5'AUGUCCUUCUCUAGGAGGU [dT][dT]3'. The Primers were designed for the respective gene expression studies and a gradient PCR was performed to standardize the optimum annealing temperature of the designed primer using 50 ng of synthesized cDNA keeping the temperature range between 50-55 oC. Caspase-2 gene-specific primer and a house keeping gene primer were validated by PCR using a mixed pool of cDNA from given cells. Primers were then validated with SYBR reactions for amplification and melt curves.
Surface conjugation of siRNA loaded nano-vesicles was carried out by layer-by-layer approach. The nano-vesicle dispersion was prepared by ethanol injection method and stabilized by polycationic polymer (Polyethyleneimine; PEI) of 1 mg/mL with molecular weight (MW) of 25 kDa. The polymeric solution of PEI was added to the pooled pellet dispersion of nano-vesicles, and the mixture was stirred for 30 min. Centrifugation at 10,000 rpm for 10 min was used to remove the extra unconjugated PEI. The resultant PEI-coated nano-vesicular dispersion was lyophilized using 2% mannitol as a cryoprotectant. The PS, ZP and PDI of the resultant mixture were determined using Malvern Zeta Sizer. The lyophilized PEI-coated nano-vesicles (1 mg/mL) were resuspended in molecular biology grade water and the resultant dispersion was added dropwise into a stirred solution of siRNA (4.22 µg/µL) and cationized HA (1 mg/mL) under aseptic condition using Laminar air flow cabinet. The resulting dispersion was stirred for 1 h before being incubated at 37 °C for 30 min. The unbound polyelectrolyte was removed by centrifugation at 3000 rpm for 10 min and lyophilized using 2% mannitol as a cryoprotectant.
Example 4: In this example, the procedure for in vitro characterization of the drug (TM) loaded nano-vesicles conjugated with DP and siRNA loaded nano-vesicles coated with targeting ligand (HA) is explained. FTIR, DSC, XRD, and HNMR analysis of plain nano-vesicles and DP conjugated nano-vesicles were performed. Particle size, polydispersity index (PDI) and zeta potential were measured using Dynamic Light Scattering using a Malvern ZetaSizer. Samples were diluted (to 10-2 weight %) in phosphate buffer pH 7.4 and measurements were reported in mean of triplicate taken at 25 °C.
Entrapment efficiency: The entrapment efficiency of TM in the nano-vesicles was determined by HPLC. The pellet formed after centrifugation at 15,000 rpm for 30 min at 4 °C was dispersed using hydration media and further lysed using organic solvent (methanol) followed by water-bath sonication for 10 min. This mixture was then diluted with phosphate buffer pH 7.4, passed through 0.22 µm syringe filter and injected into the HPLC. Entrapment efficiency was calculated as:
Entrapment efficiency (%) = (Amount of drug obtained (mg)×100)/(Initial amount of drug added (mg))
Shape and surface morphology of plain nano-vesicles and conjugated nano-vesicles was analyzed using TEM. On a copper grid that had been covered with carbon, a drop of the diluted nano-vesicle dispersion was applied, and it was given roughly two minutes to stick to the carbon substrate. Onto the carbon grid, a drop of 2% w/v phosphotungstic acid was layered, and the extra staining agent was removed using a piece of filter paper. The samples were air-dried and the ultrastructure was observed by transmission electron microscope.
Example 5: In this example, the in vitro drug release of TM from nano-vesicles in comparison to plain TM solution is described. The drug release studies were performed using dialysis method. Formulations containing TM were filled into dialysis bags (MWCO 12kDa) with both ends tightly secured. The bags were placed in 50 mL of PBS pH 7.4 under stirring at 90 rpm and at 37 ºC for 24 h. At appropriate intervals, samples were withdrawn, and an equal volume of fresh release medium was replaced. Studies were carried out in triplicate. Samples were filtered and the amount of TM present was analyzed using HPLC.
Example 6: In this example, the ex vivo corneal permeation studies of TM from plain TM solution, marketed formulation, TM nano-vesicles and DP conjugated nano-vesicles is described. Within an hour of the slaughter, the whole goat eyeball was obtained from the slaughterhouse and brought to the lab in cornisol solution kept at 4°C. To remove the adhering tissues and proteins, the cornea along with 2-4 mm of scleral tissue was rinsed with cold glutathione bicarbonate Ringer (GBR) buffer. Vertical type diffusion cells with donor and acceptor chambers were used for experiments on corneal permeability with an effective diffusion area of 0.78 cm2, and a volume of 5 mL. The isolated cornea was clamped between the diffusion cell's donor and receptor compartment with the endothelial side facing the donor cell and the epithelial side facing the latter. The receptor compartment containing 5 mL of pH 7.4 GBR buffer solution was maintained at 37±0.5 °C by flowing water in the water jacket from the thermostated water bath, and receptor solution was stirred continuously at 50 rpm using a magnetic stirrer. The formulation was placed on the top of the cornea and samples were withdrawn from the receptor compartment at various time intervals and replaced with plain GBR pH 7.4. Samples were then subjected to quantification of TM by HPLC analysis at 295 nm to determine the amount of TM in receptor solution.
Example 7: In this example, the cell line studies of the nano-vesicle formulations are described. Cytotoxicity assay: Using the MTT assay, the cell viability study was conducted on the HCE-2 and RGC-5 cell lines. Two hundred µL of HCE-2 and RGC-5 cells were individually seeded at a density of 20,000 cells/ well and allowed to grow for about 24 h in a 96-well plate. The HCE-2 cells were treated with standard TM solution, TM nano-vesicles and DP conjugated nano-vesicles at different concentration ranges (0.187-6.25 mg/mL). The RGC-5 cells were treated with standard TM solution, Hyaluronic acid-coated nano-vesicles and Hyaluronic acid-coated siRNA loaded nano-vesicles at different concentration ranges (0.187-6.25 mg/mL). The plates were incubated in 5% CO2 for 24 h at 37 °C. Following incubation, MTT reagent (0.5 mg/mL) was added after removing the spent media, and plates were incubated for 3 h. The MTT reagent was removed after 3 h of incubation, and 100 µL of solubilization solution (DMSO) was then added. Further, the absorbance of the treated and untreated cells (negative control-no treatment) was measured on an ELISA reader at 570 nm wavelength. Each treatment was carried out in triplicate and the % cell viability was calculated.
Cell uptake studies: The RGC-5 cells were used to assess the cell uptake of the formulated nano-vesicles. The cells were grown in a 6-well plate at a density of 2 x 105 cells/ 2 mL and incubated there for 24 h at 37 °C with CO2. The cells were treated with 10 µg/mL of FITC-labelled unconjugated and conjugated nano-vesicles and 1.5 µg/mL of FITC solution as control after the spent medium was drained. The cells were incubated for 24 h in 2 mL of growth media. After 24 h, all the media was removed, and the cells were then washed with Phosphate Buffer Saline (PBS). The cells were treated with 250 µL of trypsin-EDTA solution after the PBS was withdrawn, incubated at 37 °C for 3 to 4 min, and harvested into 12 x 75 mm polystyrene tubes. Finally, the tubes were centrifuged for 5 min at 300 x g at 25 °C, PBS was decanted completely and analysed by flow cytometry using the 488 nm laser for excitation and detection at 535 nm. The data obtained were further analysed using BD CellQuest Pro ver. 6.0 software.
Caspase 2 gene expression studies: The siRNAs and siRNA transfection medium (Lipofectamine 2000) were diluted in the siRNA transfection medium and reduced serum medium separately. The diluted solutions were mixed and incubated for 15-30 min at room temperature. Subsequently, the mixtures were added to each well-containing cells and transfection medium. The treated cells with only the transfection reagent were considered a control group (untreated group). The cell culture plates were incubated for 6 h at 37 °C in a CO2 incubator. Following that, DMEM containing FBS (final FBS concentration of 20%) was added, with cells being incubated under the above-mentioned conditions. To evaluate the effects of siRNAs on caspase 2 gene silencing, transfection (4×105 cells/well) was performed in 6-well cell culture plates for 48 h. The suppression of caspase 2 gene expression was then assessed by quantitative real-time PCR (qRT-PCR).
Example 8: In this example, the preclinical in vivo studies of the nano-vesicle formulation are described. Eye irritation study: Sprague Dawley (SD) rats were used to evaluate the Eye irritation as per OECD guidelines 405. Twenty-four hours before the test substance application, the eyes were observed using a slit lamp biomicroscope under white light to make sure that the eyes were free of any irritation or corneal damage. The digital photographs of the observed eye were collected for reference. For acute eye irritation study, each eye received 1-2 drops of topical ocular anaesthetic (0.5% proparacaine HCl) before the administration of the test material. For the acute eye irritation study, 0.1 mL of the test material was placed in the right eye bytightly pulling the lower lid. Each rat's left eye was used as a control. Using a slit lamp biomicroscope under white light, the eyes were examined macroscopically after the application of the test material. Digital pictures were also obtained during these examinations. The sodium fluorescein was employed for early detection of breaks in corneal epithelium and Lissamine Green was employed to examine cell degeneration or cell death using a slit lamp biomicroscope. The grades of ocular lesions were recorded by examination of digital photographs as per the Draize method. For the chronic eye irritation study, the test substance was administered 5 times a day at 5 min intervals for 1 week and observed using slit lamp biomicroscope.
Histopathological studies: The rats were sacrificed at the end of the chronic eye irritation study, and the whole eye bulbus was enucleated (surgically removed) and placed immediately in 10% Formalin solution for 48 h. The eye tissues (iris, cornea, retina, and ciliary body) were isolated and processed using alcohol and xylene. The processed tissues were embedded into molten paraffin wax mould and using microtome thin sections of tissue block were cut. The tissue sections were floated in a water bath of temperature 50-52 oC and taken on microscopic slides. Then, the tissues were stained with hematoxylin-eosin and observed under the Trinocular research microscope for any pathological changes. A MiaCam CMOS AR 6pro microscope camera and picture AR pro software was used to capture the photos.
Pharmacodynamic studies: The pharmacodynamic studies were performed using male SD rats weighing approximately 200-250 g. The right eye of each animal was the target of the experimental procedure and the left eye was maintained as normal negative control. The IOP of both was measured using iCare Home Tonometer. The animals were anaesthetized by intraperitoneal injection of xylazine (13 mg/kg) and ketamine (100 mg/kg). The IOP of the right eye was elevated by episcleral vein injection of 50 µL hypertonic saline (10% NaCl) and IOP was measured again after 30 min of glaucoma induction. The eyes were treated with nano-formulation and IOP was monitored daily for up to 1 week. Finally, the rats were sacrificed after 1 week of observation and the IOP readings were recorded using the software iCare LINK.
It will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.
, C , Claims:I/We Claim:
1. An elastic nano-vesicles composition for co-delivery of antiglaucoma agent(s) and siRNA for treatment of glaucoma, comprising:
formulation containing the mixture of two types of nano-vesicles viz., first nano-vesicles containing active agent(s) with/ without permeation enhancer, second nanovesicles containing siRNA and coated with ligand(s), with or without permeation enhancer, for the delivery of active agents, wherein the active agents are timolol maleate (TM) and siRNA, permeation enhancer is dendrimeric peptide (DP), ligand is hyaluronic acid (HA);
the dendrimeric peptide (DP) consists of branched macromolecule(s) containing peptide core and/ or peripheral peptide chains.
2. The composition as claimed in claim 1 wherein the nano-vesicle/ nanocarrier is elastic nano-vesicles composed of natural, semi-synthetic or synthetic phospholipid(s) with or without conjugation/ coating comprising of synthetic, semisynthetic or natural phospholipid(s) with or without cholesterol or any other fatty acid(s); Tween 80 or any other edge activator(s); DSPE-PEG2000-COOH or any component containing lipid; poly(ethylene imine) or any other polycationic polymer; hyaluronic acid or any other targeting agent or ligand and; any other agent(s) which enhances the performance of the formulation used in the nano-vesicle/ nanocarrier formulation/ delivery system and the quantity of these agents ranges between 0.1 to 99.9 mg, 0.1 to 50 mg, 0.1 to 50 mg, 0.1 to 25 mg, 0.1 to 20 mg, 0.01 to 25 mg and 0.1 to 50 mg respectively.
3. The composition as claimed in claim 1 and 2 wherein the nano-vesicle/ nanocarrier is selected from the class of lipidic nanoparticles, polymeric nanoparticles, metallic nanoparticles, niosomes, micelles, nano-emulsions, microemulsions, dendrimers, phospholipid-active(s) conjugates and any other nano-vesicle/ nanocarrier.
4. The composition as claimed in claim 1 to 3 wherein the peptide dendrimer may be composed of glycine, lysine, arginine, proline, histidine or other combination of amino acids thereof.
5. The composition as claimed in claim 1 to 4 wherein the active agent(s) comprising of small molecule drug, protein, peptide, nucleic acid (nucleotides, nucleosides, and analogues thereof), siRNA, shRNA, mRNA, prodrug, any other form of active agent and combination of above said active(s), which provide pharmacological action upon administration to a subject (human or non-human animal) either alone or in combination with other active or inactive components.
6. The composition as claimed in claims 1 to 5 wherein the quantity of active agent(s) ranges between 0.0001% to 60% w/v or w/w or v/v.
7. The composition as claimed in claims 1 to 6 wherein the quantity of siRNA ranges between 0.001 – 400 µg/µL or µg/µg of the final formulation.
8. The composition as claimed in claims 1 to 7 wherein the targeting ligand may comprise small molecules, proteins, peptide, antibody, carbohydrate, aptamers, nucleic acid (nucleotides, nucleosides, and analogues thereof) and any other agent, which facilitate active targeting of nanoparticulate carriers/ vesicles either alone or in combination with other active or inactive components.
9. The composition as claimed in claims 1 to 8 wherein the active agent(s) are Timolol maleate and siRNA.
10. The composition as claimed in claims 1 to 9 wherein the composition is a ophthalmic formulation used for topical/ local administration into eye, ocular or intraocular applications and may be any type of ophthalmic preparation such as eyedrops, eye lotion, eye cream, eye gel, eye ointment, eye powder, eye suspension, inserts, patches, lenses, discs, etc.
11. A method of enhancing the uptake/ release of the active agent(s) when administered by above said routes; said method involving application of composition of claims 1-10.