FIELD OF THE INVENTION

The present invention relates to a formulation of hydroxychloroquine/chloroquine along with zinc oxide nanoparticles and methods of their synthesis.

BACKGROUND OF THE INVENTION

Zinc ions have been shown to play an important role in various activities of cellular enzymes and transcription factors. Zn ²⁺ is possibly a crucial cofactor for numerous viral proteins. Enhanced intracellular zinc concentrations can inhibit RNA-dependent RNA polymerases and other proteins essential for the completion of different phases of the virus life cycle. The intracellular zinc concentration can be increased by using zinc ionophores which facilitate the zinc ions into the cells and subsequently impair the virus replication. Further, zinc also aids in upholding the strong immune responses. Zinc also exhibits anti-inflammatory property by inhibiting NF-KB signaling and modulation of regulatory T-cell functions that may limit the cytokine storm.

The versatility of zinc has been further researched in conjugation with nanomaterials. Among nanoparticle types, ZnO NPs have received high attention due to their advantage of having good biocompatibility with human cells. ZnO NPs have direct antiviral activity against many viruses, Zn has anti-viral activity as it can prevent viral entry, viral replication and spreading to organs and eventually can trigger reactive oxygen species leading to oxidative injury and viral death.

Anju Manuja et al. in International Journal of Biological Macromolecules (2021) reported microwave assisted fast fabrication of zinc/iron oxides based polymeric nanocomposites and evaluation on equine fibroblasts. The prior art document is the inventor’s own publication which discloses a novel method for preparing highly dispersible suspensions of ZnO NPs by using microwave assisted method. In contrast to those synthesized by conventional methods, microwave assisted method allowed synthesis of dispersible ZnO NPs and the incorporation of zinc/Iron oxides NPs within alginate and gum matrix (AG) in a short span of time providing high yield of the product. Further cytotoxicity and their effect on equine fibroblast cells were evaluated. Results revealed that ZnO NPs were more cytotoxic than AG ZnO NPs and doped AG Fe ₃ O ₄ doped ZnO NPs at higher concentrations.

Victor Gomes Lauriano Souza et al in Coatings (2021) reported biodegradable chitosan films with ZnO nanoparticles synthesized using food industry by- products production and characterization. This work aimed to produce bio nanocomposites of chitosan incorporated with zinc oxide nanoparticles (ZnO NPs) synthesized using food industry by-products and to characterize them. Such nanoparticles are highlighted due to their low cost, antimicrobial activity, accessibility, and sustainability synthesis. Four different levels of ZnO NPs (0, 0.5, 1.0, and 2.0% w/w of chitosan) were tested, and the bio nanocomposites were characterized in terms of their hydrophobicity, mechanical, optical, and barrier properties. Overall, the incorporation of ZnO NPs changed the composites from brittle to ductile, with enhanced elongation at break and reduced Young Modulus and tensile strength. Thus, ZnO NPs acted as plasticizer, turning the films more flexible, due to the presence of organic compounds on the NPs. This also favored permeability of oxygen and of water vapor, but the good barrier properties were maintained. Optical properties did not change statistically with the ZnO NPs incorporation.

Samy M. El-Megharbel in Coatings (2021) discloses utilizing of (zinc oxide nano- spray) for disinfection against “SARS-CoV-2” and testing its biological effectiveness on some biochemical parameters during (COVID- 19 pandemic) — ZnO nanoparticles have antiviral activity against (SARS-CoV-2). The publication reveals that zinc supplementation may increase the efficacy of hydroxychloroquine .

Chloroquine and its derivative, hydroxychloroquine, have a long history as safe and inexpensive drugs for use as prophylactic measures in malaria-endemic regions and as daily treatments for autoimmune diseases with the most common side effect being eye damage after long-term use. Chloroquine phosphate is an aminoquinoline and acts as zinc ionophore. It is a weak base which increases the endosomal pH, and is rapidly imported into acidic vesicles, and interferes with terminal glycosylation due to which the virus cannot survive. Chloroquine, through the inhibition of pH-dependent steps of viral replication, restricts human immunodeficiency virus (HIV), influenza virus, DENV, JEV, WNV infection, Zika virus.

Hydroxychloroquine (HC) is a less toxic metabolite of chloroquine and a lysosomotropic autophagy inhibitor that can be used either unaccompanied or accompanied with chemotherapy. However Hydroxychloroquine is also associated with various adverse effects, for example gastrointestinal side effects, hepatotoxicity, and blindness. Combining anti-flu medicines like lopinavir/ritonavir with chloroquine or hydroxychloroquine could cause serious arrhythmias and drug interactions. The long half-life and the enormous volume of appropriating the two medications can identify these toxic levels.

Eduardo Mansilla et al. in Cancer Biotherapy and Radiopharmaceuticals (2010) reported the lysosomotropic agent, hydroxychloroquine, delivered in a biodegradable nanoparticle system, overcomes drug resistance of p-chronic lymphocytic leukemia cells in vitro. The effect of four PEG-PLGA (polyethylene glycol-polylactic-co-glycolic acid) NP systems on drug-resistant B-chronic lymphocytic leukemia (B-CLL) cells in vitro was investigated, three of them encapsulating the drug, hydroxylchloroquine (HDQ), two with NP surface coatings of mAbs (NP1) CD20, (NP2) CD19, and CD20, and one (NP3) with no mAb, but tagged with the fluorescentmarker, fluorescein isothiocyanate. The fourth NP system(NP4) was coated with anti-CD19=FITC and anti-CD20= Alexa- Fluor antibodies, but did not contain the active drug, HCQ. The data indicates that PEG-PLGA nanoparticles with surface mAbs are suitable for selective drug delivery to B-CLL cells and produces a strong apoptotic effect when loaded with the lysosomotropic agent, HDQ (hydroxychloroquine).

Kataoka K et al. in Biomaterials (2016) reported Hydroxychloroquine-conjugated gold nanoparticles for improved siRNA activity. The impact of hydroxychloroquine conjugation on the intracellular fate and silencing activity of siRNA conjugated PEGylated gold nanoparticles was tested. Addition of hydroxychloroquine improved endosomal escape and increased siRNA guide strand distribution to the RNA induced silencing complex (RISC), both crucial obstacles to the potency of siRNA. This modification significantly improved gene down regulation in cellulo (in living cells).

Tony Y. Hu et al. in Nature Nanotechnology (2020) reported insights from nanomedicine into chloroquine efficacy against CO VID- 19. Chloroquine, an approved malaria drug is known in nanomedicine research for the investigation of nanoparticle uptake in cells, and may have potential for the treatment of COVID- 19.

Razieh Morad et al in Nature Research (2021) reported first principle simulation of coated hydroxychloroquine on Ag, Au and Pt nanoparticles. In this work, both the quantum mechanical and classical atomistic molecular dynamics approaches have been applied to demonstrate the adsorption properties of HCQ/CQ on Ag, Au, AgAu, and Pt nanoparticles. It was found that the adsorption energies of HCQ/CQ towards nanoparticles have the following trend: Pt NP>Au NP>AuAg NP>Ag NP. This shows that Pt NP has the highest affinity in comparison to the other types of nanoparticles. The (non) perturbative effects of this drug on the plasmonic absorption spectra of Ag NP and Au NP with the time-dependent density functional theory. The effect of size and composition of NPs on the coating with HCQ and CQ were obtained to propose the appropriate candidate for drug delivery. This kind of modeling could help experimental groups to find efficient and safe therapies.

Chloroquine is water-soluble and has two basic groups corresponding to the quinoline-ring nitrogen and the diethylamino side-chain nitrogen with ionization constants of 8.1 and 10.2, respectively. At a physiologic pH of 7.4, 18 % of chloroquine is monoprotonated but still soluble in lipid and able to traverse cell membranes. However, biprotonated chloroquine, as occurs in a lysosome at a pH of 4-5, is sequestered and prevented from traversing back out to the cytoplasm. Although the amount of free drug present in the plasma is miniscule at the physiologic pH, it is this form of the drug that determines the distribution of the drug between the plasma and the tissues.

There remains a need for improved formulations comprising hydroxychloroquine/chloroquine along with zinc oxide. Accordingly the present inventors have developed a nanoparticle formulation that is pH- sensitive and results in efficient delivery of zinc and hydroxychloroquine/chloroquine with or without another antiviral agent. It allows the release in an acidic milieu and which helps the Zn and hydroxychloroquine/chloroquine release inside the cell and surrounding tissues, and further enhances the bioavailability of both zinc and hydroxychloroquine/chloroquine by prolonging the duration of the drug in blood circulation with reduced cytotoxicity. OBJECTIVE OF THE INVENTION

It is thus an object of the present invention to provide a formulation comprising hydroxychloroquine/chloroquine conjugated with Zinc oxide nanoparticles having improved bioavailability of both zinc and hydroxychloroquine/chloroquine by prolonging the duration of the drug in blood circulation with reduced cytotoxicity. It is another object of the present invention to provide a formulation comprising hydroxychloroquine/chloroquine conjugated with zinc oxide nanoparticles along with a biopolymer which improves bioavailability of both zinc and hydroxychloroquine/chloroquine by prolonging the duration of the drug in blood circulation with reduced cytotoxicity.

It is yet another object of the present invention to provide a provide a method for preparing a formulation comprising hydroxychloroquine/chloroquine conjugated with zinc oxide nanoparticles along with a biopolymer, which formulation ameliorates the cytotoxicity of chloroquine.

It is a further object of the present invention to provide a formulation comprising hydroxychloroquine/chloroquine conjugated with Zinc oxide nanoparticles which delivers the hydroxychloroquine/chloroquine inside the biological tissue.

It is a further object of the present invention to provide a formulation comprising hydroxychloroquine/chloroquine conjugated with Zinc oxide nanoparticles wherein the hydroxychloroquine/chloroquine acts as a zinc ionophore.

It is further an object of the present invention to provide a formulation comprising hydroxychloroquine/chloroquine conjugated with Zinc oxide nanoparticles which acts as a lysosomotropic agent. SUMMARY OF THE INVENTION

Accordingly, the present invention provides a hydroxychloroquine/chloroquine conjugated Zinc oxide nanoparticle and the method for preparation thereof.

In an aspect of the present invention, there is provided a nanoparticle formulation comprising hydroxychloroquine/chloroquine, zinc oxide and at least one biodegradable polymer.

It is an aspect of the present invention to provide a method for synthesizing a polymeric hydroxychloroquine/chloroquine ZnO nanoparticle, wherein the method comprises the following steps: i. preparing a polymeric solution- 1% of polymer was dissolved in 1% acetic acid' w/v. 0.1% of the polymeric solution is prepared as Solution A in DW (distilled water) v/v. ii. dissolving Hydroxychloroquine/chloroquine in DMSO as solution B iii. adding 100 μM of ZnO solution in Solution A , which is further mixed with 60 pl of solution B to obtain the nanoparticles.

In an aspect of the present invention, the hydroxychloroquine/chloroquine ZnO nanoparticle formulation is effective against Buffalo Corona Virus, provides zinc ionophoric effect, provides positive lysosomotropic effect, effective against Trypanosoma evansi, and Streptococcus equi.

In an aspect of the present invention, there is provided a pharmaceutical composition comprising the hydroxychloroquine/chloroquine conjugated Zinc oxide nanoparticle and pharmaceutically acceptable additives. BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Particle size analysis of a. ZnO NPs b. CHC ZnO NPs c. YB and d.

YBHC

Figure 2: Zeta potential of (a) ZnO NP, (b) CHC ZnO NPs and (c) YBHCs

Figure 3: Scanning electron microscopic images of Zinc oxide nanoparticles (ZnO NPs), CHC ZnO NPs and YBHCs.

Figure 4: Fourier Transform Infrared Spectroscopy (FT-IR) of (a) ZnO NPs (b) HC (c) CHC ZnO NPs and (d) YBHCs

Figure 5: Cytotoxicity of (a) ZnO NPs, CHC ZnO NPs, HC and (b) ZnO NPs, YB and YBHC at different concentrations on the metabolic activity of Vero cells after 24 h incubation.

Figure 6: IC50 determined for ZnO NPs and CHC ZnO NPs.

Figure 7: Photomicrograhic images (400X) showing influence of ZnO NPs, CHC ZnO NPs, HC, YB and YBHCs on BCOV adapted vero cells.

Figure 8: Biocompatibility in SPF chick embryos A. PBS (control). B. CHCZnO NPs and C. YBHC NPs

Figure 9: Percent cell confluency of (a) ZnO NPs, CHC ZnO NPs, Untreated cells and virus control (b) YB and YBHCs.

Figure 10: Relative fold change in N gene of bCoV in infected and treated vero cells

Figure 11: Nanoparticle uptake in vero cells treated with ZnO NPs (A-C) and CHC ZnO NPs (a-c)

Figure 12: Nanoparticle uptake in vero cells treated with ZnO NPs, HC, YB and YBHCs

Figure 13: In vitro efficacy of CHC ZnO NPs against Trypanosoma evansi after 72 hours

Figure 14: In vitro efficacy of CHC ZnO NPs against Trypanosoma evansi after 24 hours

Figure 15a: Giemsa stained normal T. evansi

Figure 15b: Giemsa stained with Fluozin (i), Lysomotracker (ii and iii) Figure 16: Zone of inhibition of S. equi treated with HC, CHC ZnO NPs (Cz), ZnO NPs (3), control)

Figure 17: Microbial inhibition Control at different dilutions

DESCRIPTION OF THE INVENTION

The present invention is directed to conjugation of zinc oxide nanoparticles and hydroxychloroquine/chloroquine .

The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the present disclosure. Accordingly, it should be apparent to those skilled in the art that the following description of various embodiments of the present disclosure is provided for illustration purpose only and not for the purpose of limiting the present disclosure as defined by the appended claims and their equivalents.

It is to be understood that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component surface" includes reference to one or more of such surfaces.

Nanoparticle formulation in the present invention refers to formulations in nanoscale including the components in nanocomposite.

Zeta potential indicates the stability of nanoparticle dispersions. A high zeta potential (>|30| mV) can provide an electric repulsion to abstain the particles to agglomerate. With an increase in zeta potential, there is increase in particle surface charge also. The zeta potential value for the nanoparticles of the present invention is 200 mV, which is adequate to form a stable nanoparticle suspension. Described herein, is a nanoparticle formulation comprising hydroxychloroquine/chloroquine and ZnO nanoparticles in conjugation with at least one biodegradable polymer conjugation with zinc oxide. The present invention provides a nanoparticle formulation comprising a composite of hydroxychloroquine/chloroquine ZnO nanoparticles in conjugation with at least one biodegradable polymer.

The biodegradable polymer is a polysaccharide cationic polymer, which can easily bind and enhances transport of drug across the cell membrane. Said cationic polymer is selected from chitosan, cationic dextran, cationic cyclodextrin and combinations thereof. Preferably, the biodegradable polymer is chitosan.

The present invention thus provides a nanoparticle formulation comprising a composite of hydroxychloroquine and ZnO nanoparticles in conjugation with at least one biodegradable polymer, wherein the hydroxychloroquine/chloroquine is present in an effective amount of 0.0012-0.012 % wt, ZnO NPs is present in an effective amount of 0.05-0.75% wt and at least one biodegradable polymer is present in an effective amount of 0.01-1% wt.

The inventors have developed a nanoparticle formulation comprising hydroxychloroquine/chloroquine conjugated with Zinc oxide nanoparticles along with a biopolymer which improves bioavailability of both zinc and hydroxychloroquine/chloroquine by prolonging the duration of the drug in blood circulation with reduced cytotoxicity. It was found that the present formulation ameliorates the cytotoxicity of chloroquine.

It is a further object of the present invention to provide a formulation comprising hydroxychloroquine/chloroquine conjugated with Zinc oxide nanoparticles which delivers the hydroxychloroquine/chloroquine inside the biological tissue. The present invention also provides a method for synthesizing the nanoparticle formulation comprising hydroxychloroquine/chloroquine and zinc oxide nanoparticles in conjugation with at least one biodegradable polymer, the method comprises the steps of: i. Preparation of polymeric solution (Solution A)-

1% of polymer was dissolved in 1% acetic acid w/v. 0.1% of the polymeric solution is prepared as Solution A in DW (distilled water) v/v; ii. dissolution of Hydroxychloroquine/chloroquine in DMSO as solution B; iii. addition of 100 μM of ZnO nanoparticle in Solution A, which is further mixed with 60 pl of solution B to result the hydroxychloroquine/chloroquine ZnO nanoparticle formulation; and iiioptionally filtered through a 0.22 μM membrane and stored at 4°C.

The hydroxychloroquine/chloroquine ZnO nanoparticles of the formulation are further analyzed for size, morphology and charge. Average particle size of hydroxychloroquine/chloroquine ZnO nanoparticles was in the range of 100-1500 nm with PDI 0.282 and viscosity 0.852.

The hydroxychloroquine/chloroquine ZnO nanoparticle formulation provide a stable dispersible suspension in an aqueous solvent.

The bioavailability of hydroxychloroquine/chloroquine differs with variation in physiological pH. At a physiological pH of 7.4, 18 % of hydroxychloroquine/chloroquine is monoprotonated but still soluble in lipid and able to traverse cell membranes. However, biprotonated hydroxychloroquine/chloroquine, as occurs in a lysosome at a pH of 4-5, is sequestered and prevented from traversing back out to the cytoplasm. The inventors have found that the present nanoparticle formulation with ZnO nanoparticles, hydroxychloroquine/chloroquine in conjugation with a biodegradable cationic polymer is able to overcome the above stated problem.

The cationic polymers exhibiting pKa values between physiological pH (7.4) and lysosomal pH (4-5) can protonate at the acidic endo/lysosomal compartments. Protonation of the polymers induces an extensive influx of ions and water into the endo/lysosomes, which subsequently leads to rupture of the endo/lysosomal membrane and release the entrapped materials.

The diseased site usually has permeable blood vessels/capillaries with big fenestra so the permeation of drug-complexes into such tissues is quicker than into healthy tissues because of passive targeting and selective distribution to the diseased site.

Thus, the present invention provides a method to deliver hydroxychloroquine/chloroquine inside the biological tissues, wherein the method comprises providing a nanoparticle formulation comprising hydroxychloroquine/chloroquine, zinc oxide nanoparticles and at least one cationic polymer.

In a further embodiment of the present invention there is provided that the hydroxychloroquine/chloroquine ZnO NPs formulation can also be provided comprising at least one polymeric micelle, wherein an anionic biodegradable polymer forms a micelle with the cationic biodegradable polymer of the formulation.

It is believed that the polymeric micelle present in the formulation is stable at neutral pH but allow the fast drug release in endocytic pH. Positively charged micelles can be safeguarded by the negatively charged entities at pH 7.4 and they can be broken or unprotected at the diseased sites pH due to pH-responsive entity. Following endocytosis, the cationic entity will be protonated in the endosomal area resulting in disintegration of the polymeric micelle and destabilizing the endosomal membrane and thus support the delivery of the zinc/hydroxychloroquine/chloroquine to the cytosol.

The cytotoxic activity of hydroxychloroquine/chloroquine ZnO nanoparticles of the present invention is less compared to ZnO nanoparticles and hydroxychloroquine/chloroquine (HC) .

The cytotoxic activities of ZnO NPs, hydroxychloroquine/chloroquine ZnO nanoparticles and hydroxychloroquine/chloroquine (HC) were assessed by colorimetric assay using resazurin dye. Results revealed that ZnO NPs are more toxic than hydroxychloroquine/chloroquine ZnO nanoparticles at concentrations more than 1000 μg/ml higher.

It is hypothesized that the reduced cytotoxic activity of hydroxychloroquine/chloroquine is a result of the conjugation with zinc oxide nanoparticles, wherein hydroxychloroquine/chloroquine is in composite with at least one biodegradable polymer. The inventors have further investigated the present nanoparticle formulation for its cytotoxicity in SPF embryonated chicken eggs as represented in Figure 8. None of the embryo showed any sign of toxicity suggesting the biocompatibility of synthesized hydroxychloroquine/chloroquine ZnO nanoparticle formulation.

The inventors further found that the hydroxychloroquine/chloroquine ZnO nanoparticle formulation can also act as zinc ionophore. Said formulation being a source of Zinc ions, exhibits improved ionophoric effect. It has been shown that on treating cells with the nanoparticle formulation of the present invention, it results in an uptake of Zn ions, even though the nanoparticle formulation comprises only 0.75 % of Zn. Therefore, it is believed that the nanoparticles formulation provides zinc ionophoric effect and can be used as a source of Zinc ions in body.

The hydroxychloroquine/chloroquine ZnO nanoparticle formulation also acts as a lysosomotropic agent. Said nanoparticle formulation was evaluated against various parasites in vitro at different dilutions (1:4, 1:8, 1:10, 1:12, 1:14, 1:16 and 1:32) on daily treatment basis. It is found to be effective against various microorganisms, wherein the microorganisms such as Bovine corona virus, Trypanosoma evansi and Streptococcus equi.

Said nanoparticle formulation can be used as alternative drug substances (API) with an improved toxicity profile compared to hydroxychloroquine/chloroquine. Accordingly, in an embodiment of the present invention, there is provided that a pharmaceutical composition comprising the nanoparticle formulation of the present invention. Said pharmaceutical composition comprising nanoparticle formulation can be used a medicament.

The medicament may be a pharmaceutical composition or pharmaceutical dosage form comprising nanoparticle formulation of the present invention. Suitable pharmaceutical dosage forms include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, tablets, capsules and pills. The pharmaceutical dosage forms facilitate administration of nanoparticle formulation to a mammal, preferably to a human and animal.

The pharmaceutical dosage forms may further comprise one or more pharmaceutically acceptable additives, such as binders, carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients. Additional antiviral agents can be combined with the present nanoparticle formulation.

The present invention is now being illustrated by way of non-limiting examples.

Examples

Raw Materials

Chitosan (CS: deacetylated chitin, 75-85% deacetylation), were purchased from Sigma-Aldrich Chemicals Private Ltd. (Bangalore, India). Zinc sulphate was procured from Qualigens Fine Chemicals Pvt. Ltd., Mumbai, India. Yasad bhasam was purchased from Baidyanath, India. Hydroxychloroquine was purchased from Ipca laboratories, India. The FluoZin-3 AM probe and LysoTracker probe were purchased from Life Technologies Co. (Carlsbad, CA).

Other chemicals and reagents mentioned in this study were also of analytical grade and purchased from Sigma-Aldrich Chemicals Private Ltd. (Bangalore). The cell lines, primary cell cultures were maintained in DMEM supplemented with penicillin (100 units/ml), streptomycin (100 mg/ml) and 10 % FBS. The cells were incubated at 37°C in a humidified, 5% CO2 atmosphere. All glassware used in this study was from M/S Borosil (India).

Example 1: Synthesis of zinc oxide nanoparticles

Dispersible ZnO NPs was synthesized via pulse on and off microwave-assisted rapid method using zinc sulfate as a precursor. Patent application number 201911009696 describes such process and is incorporated herein by reference. For the synthesis of ZnO NPs using zinc sulfate and sodium hydroxide solutions in distilled water, sodium hydroxide solution (4M) solution A in 1 mL quantity was added to solution B zinc sulfate solution (IM) in a ratio of 1: 40. ZnO NPs as white precipitate was collected by centrifugation or microwave irradiation at 600 watts with pulse on and off (3 min on and 2 min off), subsequent washings with 70% ethanol and water, microwave the reaction mixture using microwave with pulse on and off to reduce the risk of overheating the solvent.

Example 2: Synthesis of hydroxychloroquine/chloroquine ZnO nanoparticles loaded with polymeric composites

Polymeric solution was prepared by mixing 1% of chitosan in 1% acetic acid' w/v. Further, 0.1% of the polymeric solution was dissolved in DW (distilled water) v/v to prepare Solution A. Hydroxychloroquine 2mg/ml was dissolved in DMSO w/v as solution B. Then 100 μM of ZnO nanoparticle was added in Solution A, which is further mixed with 60 μl of solution B to obtain the desired nanoparticle formulation, which optionally can be filtered through a 0.22 μM membrane and stored at 4°C.

Example 3: Synthesis of Yasad bhasam (YB) and hydroxychloroquine/chloroquine (HC) blended composites (YBHC)

Yasad bhasma is a zinc-based ayurvedic preparation in which zinc based nanomedicines can be developed. A nanoformulation using hydroxychloroquine/chloroquine and zinc based Ayurvedic Yasad bhasam which includes zinc and traces of Fe ions, was prepared. The nanoformulation does not comprise any biodegradable polymer.

Yasad bhasam 75 mg was dissolved in 10 ml distilled water to form YB solution. 60 μl of hydroxychloroquine/chloroquine (2mg/ml DMSO) added in 10 ml of YB solution v/v and mixed thoroughly and filtered through a 0.22 μM membrane and stored at 4°C to produce a YBHC nanoformulation.

All the formulations were characterized for size, morphology, surface charge, interaction of various chemicals used during synthesis. The formulations were also evaluated for cytotoxicity, their efficacy against buffalo corona virus and also for the lysosomotropic action on buffalo corona virus adapted vero cells. Size, Morphology and Charge

Dynamic light scattering (DLS) was used to measure the average particle size and size distribution (polydispersity index) of formulated ZnO NPs, CHC ZnO NPs and YBHC at 25°C using the Zetasizer nano ZS (Malvern/Microtracy instruments). The zeta potentials of the same samples were determined by the laser light scattering technique. It can make a prediction about the stability of colloid dispersions. The zeta potential measurements are performed by using an aqueous dip cell in an automatic mode. Samples are diluted in distilled water and placed in the capillary measurement cell, with the cell position adjusted.

The morphology of the ZnO NPs, CHC ZnO NPs and YBHCs were determined by scanning electron microscopy (SEM, Hitachi S-3400N) with EDAX detector (AMETEK, USA) and used for imaging and element detection of samples in the present study. The samples for SEM were mounted on metal stubs with adhesive tapes and recorded at an accelerating voltage of 13 KV. Transmissible electron microscopy was also used to study the morphology of ZnO NPs.

The Zinc oxide nanoparticles were observed in the shape of flowers as determined by SEM (Figure la). The particle diameter (z-average) taken by zetasizer was less than lOnm with 0.0 polydispersity index (PDI) indicating mono dispersion (Figure lb) of the ZnO particles.

The PDI index of ZnO NPs ranges from 0 (monodisperse) to 1.0 (very broad distribution).

The particle size of CHC ZnO NPs was in the range of 100-1500 nm with PDI 0.282 and viscosity 0.852.

The particle size of the YB was shown as microparticles whereas the YBHC showed majority of the particles from 1 to 100 nm range (Figure 1c and Id) and hence is a nanoformulation. YB were mostly observed as microparticles with PDI 0.0158 suggesting better dispersion, while YBHC being a nanoformulation is slightly less dispersible as compared to YB.

It is noteworthy that the hydrodynamic diameter of the particles measured by dynamic light scattering is higher than the size estimated from electron microscopy particularly because light scattering studies gives a volume distribution weighing especially large particles and chitosan nanoparticles have a high swelling capacity. It is therefore highly sensitive to detect a few larger particles resulting from particle aggregation during storage of the suspensions.

Zeta Potential

Zeta potential provides a prediction about the stability of nanoparticle dispersions. A high zeta potential (>|30| mV) can provide an electric repulsion to abstain the particles to agglomerate. With an increase in zeta potential, there is increase in particle surface charge also. The zeta potential values (200 mV) are adequate to form a stable nanoparticle suspension. In case of charged particles; the repulsive interactions will be larger between the particles as the zeta potential increases, leading to the formation of more stable suspension with a more uniform size distribution.

The zeta potential of ZnO NPs, CHC ZnO NPs was 200mV (Figure 2), while for YBHC it is 0.2mV. ZnO NPs showed negative polarity, whereas the polarity of CHC ZnO NPs remains undetermined. It may be due to combination of cationic chitosan and anionic ZnO NPs. However, the polarity of YB and YBHC is positive.

Figure 3 shows the scanning electron microscopic images of Zinc oxide nanoparticles (ZnO NPs), CHC ZnO NPs and YBHC. The Zinc oxide nanoparticles were observed in the shape of flowers as determined by SEM (Figure 3 a and TEM (Figure 3b).

Elemental quantification

Table 1 shows the elemental quantification of ZnO NPs, CHC ZnO NPs and YBHC as obtained by Energy dispersive X-ray spectra (EDS) of the nanoparticles.

The concentration of Zn was observed as 43.00% 33.79% and 73.37% in ZnO NPs, CHC ZnO NPs and YBHCs respectively whereas as ‘O’ was observed higher in CHC ZnO NPs, followed by ZnO NPs and then YBHCs. ‘S’ was observed in ZnO NPs and CHC ZnO NPs due to zinc sulphate used in synthesis of NPs, while it was not observed in YBHCs. Similarly ‘Fe’ was observed only in YBHC.

Fourier Transform Infrared Spectroscopy

Fourier Transform Infrared Spectroscopy (FT-IR) of ZnO NPs, CHC ZnO NPs and YBHCs was performed to see the interaction of various chemicals used during synthesis. The FTIR spectra of ZnO NPs, Hydrochloroquine, CHC ZnO NPs and YBHC are shown in Figure 4 (a-d). The FTIR spectrum of ZnO NPs recorded in the range of 400 to 4000 cm ^-1 . Characteristic peaks of chitosan are located at 3444.89 cm ¹ for hydroxyl group and 1521.84 cm ^-1 for the amino group. Carbonyl stretching of remaining acetamide group in chitosan is observed by the peak at 1645.28 cm ^-1 . The aromatic C-H stretching of HC was observed at 2917.51 cm-1.The -OH stretching frequency appeared at 3223.87 cm-1 of hydroxychloroquineis shifted to 3299.17 cm-1 in the CHC ZnO NPs. The aromatic C=C stretching in two regions 1613.06 cm-1 and 1457.70 cm-1 in HC sample is shifted to 1616.77 cm-1 and 1508.33 cm-1 respectively in CHC ZnO NPs. Similarly, the C-Cl stretching frequency at 1033.85 cm-1 also showed a marked shift to 1019.96 cm-1 in the solid inclusion complex. The C-N bending frequency at 1114.57 cm ^-1 in the HC is shifted to 1157.29 cm ^-1 in the CHC ZnO NPs. The FTIR spectra of YB is generally pure and very less peaks are observed, the very small amount of HC blended in YB does not show marked shifts of peak. The marked changes in the FTIR spectra of ZnO NPs/HC/CHC ZnO NPs/YBHC indicate both the molecules are included in the formulation.

Example 4: Cytotoxicity studies on African green monkey kidney cells (Vero cells)

The cytotoxic activities of ZnO NPs, CHC ZnO NPs, HC, YB, and YBHC were assessed by colorimetric assay using resazurin dye. Results revealed that ZnO NPs were more toxic than CHC ZnO and HC, followed by YB, YBHCs at concentrations more than 1000 μg/ml (Figure 5(a)-(b)). The difference in toxic effects between concentrations at higher concentrations was found to be statistically significant (10 μg/ml and 5 μg/ml, p< 0.05). Insignificant percentage of cytotoxicity at lower concentrations was observed. The mechanism regarding the cytotoxicity of ZnO NPs was primarily by production of reactive oxygen species. These data were plotted and Hill 4 parameter sigmoidal regression was performed on AAT BioQuest. IC 50 calculated for ZnO NPs was 639 and for CHC ZnO NPs was observed as 734 (Figure 6). Example 5: Biocompatability in SPF chick embryos

The early stages of chickens’ rapid embryonic growth provide a sensitive model for investigating toxicity. SPF embryonated chicken eggs, in triplicates, were inoculated with two selected dilutions of CHC ZnO NPs (1:4 and 1:8) and YBHCs (1 :4) per egg in 100 μl quantity through allantoic route. The eggs were visualized daily for up to 5 ^th day post-inoculation for mortality of the embryos. None of the embryo showed any sign of toxicity suggesting the biocompatibility of synthesized CHC ZnO NPs and YBHCs (Figure 7).

Example 6: Virus propagation in African green monkey kidney cells (Vero cells)

Buffalo Coma virus strain, BCoV isolated from a diarrheic buffalo calf from Haryana, India, and propagated in Vero cells. Vero cells, in triplicates were infected with BCoV for one hour at 37°C and the cells were then washed with PBS and the cell lysates were prepared by rapid freeze-thaw method.

Example 7: Effects of formulations on African green monkey kidney cells (Vero cells)

Vero cells, in triplicates were infected with bCoV for one hour at 37°C and the cells were then washed thrice with PBS. The virus infected vero cells were treated with ZnO NPs, CHC ZnO NPs, HC, YB and YBHCs in two fold dilutions (2000 to 62.5 μg/ml); Untreated and uninfected cells were kept as negative control, whereas and virus infected cells without treatment were kept as positive control. The cell morphology of formulation CHC ZnO NPS, YBHC and YB treated cells and untreated uninfected controls were in good conformity and showed better proliferation and monolayer formation (Figure8) with confluence 100% (Figure 9) followed by ZnO NPs (80%) (Table 2). Cell treated with HC were deformed, aggregated and degenerated depicted some morphological/detrimental effects on the cells. Virus control after 24 hrs started showing cytopathic effects (CPE) like rounding of cells and after 72 hrs of post treatment CPE rounding and desquamation of cells is clearly visible. No CPE was observed on the cells treated with any of the formulations as well as untreated one.

Table 2: The cell confhiency (%) of formulation CHCZnO NPS, YBHC and YB treated cells and untreated uninfected controls

Example 8: Buffalo Corona virus infected Vero cells exhibited M protein

It was intended to determine whether the vero cells have adapted the BCoV after the infection. RNA was extracted from BCoV infected cells treated with different formulations as well as untreated control, transcribed by reverse transcription and amplified M protein as per the details given earlier in methodology section. The M protein, the most abundant viral protein is responsible for providing a specific shape to the viral envelope; binding to the nucleocapsid and organizing coronavirus assembly. Coronavirus M proteins are highly diverse in amino acid contents but maintain overall structural similarity within different genera. The viral scaffold is maintained by M-M interaction. Of note, the M protein of SARS- CoV-2 does not have an amino acid substitution compared to that of SARS.

The positive control (Only virus infected samples), treated samples yielded 523 bp product specific to M protein. The amplification in negative control (uninfected and untreated samples) was not observed. The representative samples sent for sequencing further confirm the bovine corona virus specific M protein and submitted to GenBank NCBI and assigned a GenBank accession number MW592691 to this sequence. No results were found in supernatants indicating either no replication of virus or it is too little to detect suggesting the efficacious formulations against the virus.

Further the levels of bCOV were quantitated in the vero infected cells by quantitative real-time PCR to determine relative fold change in N gene of bCoV. CHC ZnO NPs able to inhibit 6.8 fold times N gene as compared to controls, whereas HC could inhibit only two times (Figure 10).

Example 9: Inhibitory action of CHC ZnO NPs

Further the ability of formulations to inhibit the virus before infecting the vero cells was explored. The virus was treated with different concentrations of formulations and observes how the treated virus can infect the vero cells. Cell proliferation was determined visually as well as by calorimetric method using resazurin dye after normalizing with the background.

No good proliferation was observed at 1:4 dilutions of CHC ZnO NPs, since it was toxic to the cells along with the virus. However, 1:8 and 1:16 dilutions exhibited good cell proliferation.

Example 10: Nanoparticle uptake in vero cells

Chloroquine being a well known lysosomal targeting agent, the intracellular zinc distribution was examined after treatment of Vero cells with treated with ZnO NPs, HC, CHC ZnO NPs YB, and YBHCs. As shown in Figure 11, ZnO NPs (Figure 11 A, B and C) and CHC ZnO NPs (Figure 11 a, b and c) treated cells showed accumulation of intracellular zinc ions primarily in the lysosomes, as evidenced by co-localization of the fluorescent signals of FluoZin-3 and LysoTracker. However, no fluorescence or Zinc internalization was observed in HC, YB or YBHC treated cells (Figure 12). It indicates that CHC ZnO NPs formulation of dispersible zinc oxide exhibit lysosomotropic effect with or without chloroquine. Vero cells also uptake Zn ions from CHC ZnO NPs which contains only 0.75 % of ZnO NPs. The cell proliferation in YBHC treated cells was the highest amongst other treatments. It indicates these formulations do not exhibit internalization or lysosomotropic effect, indicating some other factors facilitating cell proliferation. At the same time, these nanoformulations are showed better biocompatibility than others in terms of cell proliferation, even better than untreated control.

Therefore, in an embodiment of the present invention it is provided that the hydroxychloroquine/chloroquine ZnO nanoparticle provides lysomotropic effect, wherein the nanoparticles are in conjugation with at least one biodegradable polymer.

In conclusion, since both the hydroxychloroquine/chloroquine and zinc are considered potential components for the management of viral infections, the efficient delivery of combination of the two can be considered as a novel strategy to develop more effective approaches.

Example 11: In vitro efficacy of CHC ZnO NPs against Trypanosoma evansi

Formulated CHCZnO NPs were evaluated for in vitro growth inhibitory efficacy on T. evansi parasites (lx 10 ⁶ /ml) in vitro at different dilutions (1:4, 1:8, 1:10, 1:12, 1:14, 1:16 and 1:32) on daily treatment basis. CHC ZnO NPs contains 7.5mg/ml of ZnO NPs. At 24 hrs, the 100% parasites were killed at the concentration starting from 1:4 to 1:14, reduced at concentration 1:16 followed by 1: 32. However, after 48 hrs, live parasites could not be demonstrated in 1:16 dilution. After 72 hrs, complete growth inhibition was observed at all the concentrations in wet smears (Figure 13 and 14). Whereas the untreated T. evansi parasites exhibited continuous increase due to dividing parasites (Table 3a and Table 3b). Giemsa staining of normal T. evansi (Figure 15a i, ii) stained with Fluozin, Lysomotracker [Figure 15 bi, bii and biii) are also shown. The staining with fluozin revealed that ZnO NPs is present on the surface of the parasite.

Table 3: In vitro efficacy of CHC ZnO NPs against Trypanosoma evansi a. WET SMEAR b. Thin smear Example 12: Effect of CHC ZnO NPs on Streptococcus equi

This antibacterial activity of ZnO NPs, HC and CHC ZnO NPs was determined against Streptococcus equi. Nutrient agar 2.8 gm was dissolved in 100 ml of distilled water and autoclaved at 121°C at 15 psi for 15 min(s). Petri dishes were poured with 20 ml of agar to maintain 3-4 mm thickness and kept for 15 min(s) for solidification. All the petri dishes were kept in an incubator for 24 hr(s) in order to check the sterility. After 24 hr(s) they were streaked with 0.5 McFarland standard of S. equi culture and hollow of 10 mm diameter wells were cut by using sterile gel cutter.10 mg of ZnO NPs, HC, CHC ZnO NPs were dissolved in 1 ml of distilled water separately and all the tubes were vortexed and additionally sonicated for 20min to avoid aggregation of particles. Sixty pl was added to the well and kept for 2 hr(s) for better diffusion. Petri dishes were placed with inverted position in the incubator at 37°C for 24 hr(s). After incubation, a zone of inhibition was measured in mm along with the control. (Figure 16)

Various dilutions are prepared ranging from 10 mg/ml to 0.6 mg/ml.

Example 13: Microbial Inhibitory Concentration

This antibacterial activity of ZnO NPs, HC, and CHC ZnO NPs against S. equi was studied quantitatively through MIC Broth dilution method. The further analysis of antibacterial tests has been conducted through obtaining single colonies and their further inoculation in experimental broth. They are incubated at 37 °C for 24 hours in a B.O.D incubator. The nanoparticle formulations are further diluted and dispersed into distilled water in various amounts ranging from 1000g/mL- 15.62 μg/ml. They are further incubated along with Negative and Positive control for 24 hours and their absorbance is measured at 600nm using ELISA plate reader, Power Wave XS2 (Figure 17). ADVANTAGE OF THE INVENTION

The present invention therefore provides a formulation comprising hydroxychloroquine/chloroquine and dispersible ZnO NPs in conjugation with a cationic biodegradable polymer. The formulation reduces the toxicity of hydroxychloroquine/chloroquine and provides a safe way to deliver hydroxychloroquine/chloroquine inside the biological tissue at lysosomal pH. Due to ionophoric effect of hydroxychloroquine/chloroquine the formulation can also be used as source of zinc ions in the body. Despite of benefits of the formulation, the formulation is easy to prepare and does not require costly equipments or chemicals.