WO2016128988A1 - A process for the preparation of polyacryloyl hydrazide stabilized metal nano particles and the products obtained thereby - Google Patents

A process for the preparation of polyacryloyl hydrazide stabilized metal nano particles and the products obtained thereby Download PDF

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WO2016128988A1
WO2016128988A1 PCT/IN2015/000383 IN2015000383W WO2016128988A1 WO 2016128988 A1 WO2016128988 A1 WO 2016128988A1 IN 2015000383 W IN2015000383 W IN 2015000383W WO 2016128988 A1 WO2016128988 A1 WO 2016128988A1
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pah
hydrazide
nano particles
nps
polyacryloyl
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PCT/IN2015/000383
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French (fr)
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Umaprasana Ojha
Rewati Raman UJJWAL
Anuj Kumar
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Rajiv Gandhi Institute Of Petroleum Technology, Rae Bareli
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    • A—HUMAN NECESSITIES
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
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    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue

Definitions

  • This invention relates to an one step process for preparing metal nano particles such as silver or gold nano-particles and in particular to a polyacryloyl hydrazide (PAH) stabilized silver (Ag) or gold (Au) nano particles (PAH-Ag/Au NPs) suitable for biomedical and drug delivery applications.
  • PAH polyacryloyl hydrazide
  • This invention further relates to an one pot synthesis of drug loaded polyacryloyl hydrazide stabilized silver/gold nano particles from the above PAH-Ag/Au NPs and the product obtained thereby as well as to a process of synthesis of a hydrogel based sustained release formulation thereof.
  • the Ag/Au NPs of the invention has controlled and narrow size distribution.
  • Nano particles are of great scientific interest as they are, in effect, a bridge between bulk materials and atomic or molecular structures.
  • the properties of materials change as their size decreases to the nano scale and thereby the percentage of atoms at the surface of the material becoming significantly higher. This reduction in size leads to many interesting and sometimes unexpected properties.
  • Silver nano particles are nano particles of silver.
  • the nano particles in respect of silver are with a particle size generally between 1 nm to 1000 nm in diameter in average, more specifically between 1-100 nm in diameter in average.
  • a plurality of particle diameters or sizes exist in the nano particles.
  • great emphasis is given to the narrow range of relatively smaller particle sizes as well the shape of the nano particles for various applications.
  • Ag NPs silver nanoparticles
  • Synthesis and surface grafting of silver nanoparticles have gathered huge interest in recent times due to their useful antimicrobial properties, catalytic activities in the form of a reducing/oxidizing agent, electrical and thermal conductivity and optical properties.
  • Ag NPs have found applications as sensors, catalysts in redox reactions, electronic devices and antimicrobial agents.
  • the biocidal activities of bare Ag NPs though interesting, is associated with potential toxicity towards cells due to release of Ag + cations from Ag NPs. Therefore, efforts were made to immobilize Ag NP into a biocompatible material, typically a polymer matrix to resist oxidation (cf. AshaRani, P. V.; Mun, G. L.
  • polymeric capping agents were preferred over their low molecular weight analogs such as sodium citrate, since the later is known to possess unfavorable anticoagulant activity (cf. Ocwieja, M.; Adamczyk, Z. Langmuir 2013, 29, 3546-3555).
  • the polymer matrix further helped by releasing the Ag NPs in a controlled manner to achieve desired bactericidal effect and avoid eukaryotic toxicity (cf. Zhu, Y.; Morisato, K.; Li, W.; Kanamori, K.; Nakanishi, K. ACS Appl. Mater. Interfaces 2013, 5, 2118-2125 & Liu, J. Y.; Sonshine, D. A.; Shervani, S.; Hurt, R. H. ACS Nano 2010, 4, 6903-6913).
  • these publication does not disclose any hydrazide functionality but glutathione, cysteine etc.
  • Another problematic aspect of traditional Ag NP synthetic procedure is the use of inorganic or organic reducing agents.
  • the biocompatibility of the resulting Ag NPs containing trace amount of these toxic impurities is questionable. Therefore, the focus has shifted to the development of benign and green procedures involving polymeric reagents to synthesize the NPs for various biomedical applications.
  • polyamines were used as nanoreactors to synthesize NPs due to their efficient capping ability (cf. Dai, J.; Bruening, M. L.
  • hydrazine and/or substituted hydrazine was envisaged because of hydrazines having solubility in water, polar or non-polar organic solvents depending on the substitution; having strong to weak reducing ability depending on the substitution; and nonexistence of non-volatile metal ions as in other reducing agents such as, for example, sodium borohydride, which would facilitate the removal of by-product or unreacted reducing agent.
  • the above publication discloses a four step process containing adding a first amount of organoamine in a solvent followed by addition of silver salt, a second amount of organoamine and an organohydrazine in different weight ratios and in different temperature conditions. Therefore such process becomes much complicated. Further biocompatibility of the different organoamines for medical applications is not confirmed and the silver nano particles produced by the above process is mainly used in the manufacturing of thin film transistors.
  • Patent publication US 20120225126 discloses a solid state polymer stabilized Ag NP synthesizing process.
  • this publication requires a mechanical milling process and the polymers used for the process such as polyvinyl pyrrolidone (PVP) may not be suitable for internal drug delivery applications.
  • PVP polyvinyl pyrrolidone
  • the publication does not teach any method to overcome the complexity of the reaction process associated with organic polyamines and hydrazides rather it teaches away towards a solid state process to avoid the complexity of a liquid state process for the preparation of Ag NPs.
  • ligand stabilized Au nano particles are reported to have been prepared from Au m chlorides and some of which can be used in drug delivery complexes.
  • US20140186263 discloses a maleimide-functionalized gold nanoparticle comprising a ligand monolayer, wherein said ligand monolayer includes at least a PEG-thiolated ligand.. HAuCI 4 i.e.
  • AuCI 3 in acidulated water is capped with polyethylene glycol and thereafter made to react with a furan-protected maleimide-PEG-thiol to produce a furan-protected maleimide-functionalized gold nanoparticle, followed by heating the furan-protected maleimide-functionalized gold nanoparticle intermediate under conditions suitable for removal of the furan-protection group and to provide the maleimide-functionalized gold nanoparticles. Therefore, the preparation process including the reactants disclosed in the specification is quite complex.
  • Gold 1 halides such as AuCI and AuBr are seldom used to prepare gold nanoparticles but because of their non-stable character, a Iky I amines such as oleylamine or octadecylamine can be used for reduce the gold halides to gold nanoparticles c.f. Lu et al; PMC 2008; Chemistry. 2008; 14(5): 1584-1591.
  • the AuNPs synthesized through this procedure are bare NPs. These NPs cannot be used to load drug for targeted delivery application. These NPs also lack nanogel formation ability. Further, the use of low molecular weight amine as reducing agents, the resultant gold nano particles are not useful for biomedical applications due to toxicity issues associated oleylamine or octadecylamine.
  • It is yet further object of the invention is to provide a simple process for preparing a drug loaded polyamine/polyhydrazide stabilized gold nano particles trapped in a hydrogel for slow release formulation.
  • the present invention provides a process for preparation of polyacryloyi hydrazide (PAH) stabilized metal nano particles comprising reacting aqueous solution of an a cry I oy I hydrazide polymer with a suitable metal salt solution in ambient conditions.
  • PAH polyacryloyi hydrazide
  • the invention further provides a process for preparation of drug loaded polyacryloyi hydrazide stabilized metal nano particles comprising
  • the invention also provides a process for preparation of a sustained release formulation of drug loaded polyacryloyi hydrazide stabilized metal nano particles comprising
  • Said metal nano particles of the invention are acryloyi hydrazide (PAH) homo or copolymer stabilized siver or gold nano particles.
  • PAH acryloyi hydrazide
  • Non limiting examples of such polymer are PAH or PAH-co-polyethylene glycolacrylate or PAH-block-polyethylene glycolmethylacrylate (PAH-PEG MA).
  • Said metal salt is silver nitrate or aurum chloride at a preferred concentration in the range of 0.02 mmol/l to 4 mmoL/l and more preferably 0.2 mmol/l.
  • concentration of said aqueous solution of PAH is preferably 0.02 g/ml.
  • Said PAH-Ag nano particles have an average particle diameter in the range of 8-141 nm and preferably between 20- 30 nm.
  • the average particle diameter of the metal nano particles is controllable by controlling the concentration of said metal solution and/or by controlling the pH of the reaction mixture.
  • the invention further provides a polyacryloyi hydrazide stabilized pH responsive silver or gold nano particle and a polyacryloyi hydrazide stabilized pH responsive silver or gold nano particles.
  • the invention also provides an antitumor drug loaded polyacryloyi hydrazide stabilized silver or gold nanoparticles and an hydrogel based slow release formulation of drug loaded polyacryloyi hydrazide stabilized silver or gold nanoparticles.
  • said hydrogel forming agent is selected from acrylic acid, dimethyl 2,2'- dithioacetic acid or dimethyl malonate and preferably the drug is selected from camtothecin, doxorubicin, taxol, paclitaxel, everolimus etc.
  • Figure 1 is a schematic illustration of PAH-Ag NPs formation and drug encapsulation followed by pH dependent controlled release of the drug and silver nano particles according to the invention.
  • A indicates coloration of the PAH-Ag NPs of the invention under different AgN0 3 concentration
  • C is DLS traces of PAH-Ag NPs synthesized using different concentrations (0.2 - 4.0 mmol/l) of AgN0 3 and 2 wt% solution of PAH.
  • Figures 3 A-F show the HRTEM images of PAH-Ag NPs synthesized using
  • B is the particle size distribution for "A"
  • F is the particle size distribution for "E"
  • H shows the diffraction pattern of a PAH-Ag NP synthesized using 4.0 mmol/l of AgN03 from HRTEM.
  • A is the visual observation of NP formation, drug loading and hydrogel formation stages;
  • B is the UV-Vis spectra of the PAH-Ag NP and drug loaded PAH-Ag NP;
  • E is the fluorescence microscopic images of intracellular uptake of PAH-Ag-RITC NPs in MCF-7 cells.
  • Figure 5 shows the FT-IR spectrum of the lyophilized NPs of the invention.
  • Figure 6 is the absorbance profile of aqueous NPs of the present invention.
  • Figure 7 represents the average particle size variation of the NPs of the present invention vis-a vis pH of the reaction mixture, which is a characteristic behavior of pH responsive nanogel.
  • Figure 8 is the release profile of CPT at pH 5.4 from PAH-Ag-CPT NPs of the present invention.
  • Figure 9A-9C show: (A) Color and (B) UV-Vis traces and (C) DLS traces of PAH-PEGMA capped Ag NPs synthesized using various concentration of AgN0 3
  • Figure lOA-lOC show: (A) Color and (B) UV-Vis traces of the PAH-Au NPs synthesized using various concentration of AuCI and (C) Absorption maximum versus [AuCI] plot of PAH-Au NPs of varying particle size.
  • Figure 10D shows DLS traces of PAH-Au NPS synthesized using varying concentration of AuCI.
  • Example of such polymers includes but is not limited to polyacryloyl hydrazide
  • PAH PAH-block-polyethylene glycolmethylacrylate
  • PAH-PEGMA PAH-block-polyethylene glycolmethylacrylate
  • Polymers of acryloyl hydrazide can be represented by a general structure of formula 0)
  • Ri and R 2 are H or CH 3 , in a preferred embodiment Ri is CH 3 and R 2 is H; R3 is H or a linear or branched CpH 2 p + i (p is a natural number), in a preferred embodiment R3 is H;
  • R 4 may be an oligo or polymeric polyethylene glycol moiety, having a Molecular Weight of 300-5000, typically 500.
  • n can be 0 or a +ve integer and m can be any number in between 10-10000.
  • Exemplary structures of the polymers can have a molecular structure of formula (ii) and (iii);
  • PAHs for the synthesis of metal NPs are found to be multifold.
  • Polyacryloyl hydrazide homo or copolymers possess hydrazide functionality in each repeating unit.
  • the presence of large amount of carbazide groups makes PAHs a stronger reducing agent for the metal ion precursors in comparison to that of normal polyamines.
  • the -NH2 group present in the pendant hydrazide moiety can swiftly cap the surface and stabilize the resulting NPs. Presence of hydrophilic PAH on the surface of metal NPs would assist easy dispersibility in aqueous media.
  • PAH pH responsiveness of PAH can be swiftly utilized to encapsulate various drug molecules in the NPs and to release them in a pH controlled manner under physiological conditions.
  • PAH also forms cytocompatible, injectable and stimuli responsive hydrogels with a range of crosslinkers. Therefore, the procedure may be extended to encapsulate the synthesized Ag/Au NPs along with drug molecules in-situ through formation of hydrogels.
  • PAH-Ag/Au NPs formed by the process of the invention has controlled and narrow range of particle size as well as it can be formed with a predominantly predetermined shape which are important aspects of preparing the biomedical drug delivery complexes.
  • FIG. 1 is a schematic illustration summarizing the whole process as would be described below.
  • the inventive PAH-Ag NPs has the added advantage of encapsulating and releasing drugs in a controlled manner.
  • PAH is soluble is water, but a polar solvent such as DMF, NMP etc may also be used.
  • a polar solvent such as DMF, NMP etc may also be used.
  • silver nitrate is used as the silver salt but other salts such as silver halides, sulfates, acetates may also be used for the above reaction.
  • the Ag NPs are synthesized by adding different concentrations of aqueous AgN0 3 to 0.02 gm/ml aqueous solution of PAH (pH «8.7) at room temperature.
  • the hydrazide functionality present in PAH served as both reducing agent for AgN03 and surface capping agent of the resulting NPs.
  • the formation of Ag NPs has been swift and the reaction is completed in about 5-20 minutes under ambient conditions and a characteristic yellow color (cf. Figure 2A) of the reaction mixture appears within around 15 minutes of the addition of 0.2 mmol/l AgN03.
  • Even lesser concentration of AgN0 3/ i.e. upto 0.02 mmol/l is quite effective in producing PAH stabilized silver nano particles with desired characteristics.
  • 0.2 mmol/l of AgN0 3 is the most desirable concentration. This is schematically shown in Figure 1 wherein (i) shows the _CONHNH 2 moiety and PAH- AgNPs are shown in (ii).
  • the resulting PAH-AgNPs can be purified by diaiyzing the reaction product thus formed.
  • PAH may be used in excess rather than AgN0 3 so that toxicity associated with Ag + ion can be avoided. Any unreacted PAH may be removed in this stage by way diaiyzing and/or centrifuging the reaction mixture. The reaction being quantitative carefully executed one completely eliminates the possibility of any uncapped metal NPs and the need for separation of resulting mixture becomes minimal. PAH is attached with the surface of the silver nano particles and is considerably stable.
  • the silver nano particles and PAH form the complex through chemical bond and/or other physical attachment such as covalent bonding, hydrogen bonding, coordination complex bonding, ionic bonding or a mixture of different chemical bonds, while the physical attachment may be through van der Waals' forces, dipole- dipole interaction or a mixture of different physical attachments.
  • Table 1 shows the particle size, yield and surface charge of Ag NPs produced by the inventive process in different AgNC>3 concentrations , wherein *the FWHM is determined from UV-Vis spectroscopic analysis, "the wt% of Ag in PAH-Ag NP is determined from AAS analysis, *the average particle diameter (d avg ), PDI and Zeta potential ( ⁇ ) are determined from DLS analysis.
  • the Ag NPs synthesized using 0.2 mmol/l of AgN0 3 are mostly spherical and the particle diameters are in the range of 8-35 nm.
  • Form Figure 3B it can be seen that the size distribution is considerably narrow, with the particles possessing diameters in the range of 20 - 30 nm are the most abundant ones.
  • FESEM images also displayed clusters of NPs possessing size distribution similar to that of the HRTEM data. It is seen that the average size of NPs increases with the increase in the amount of AgN0 3 in the reaction mixture.
  • the average size of the PAH-Ag NPs synthesized using 0.5 and 2.2 mmol/l of AgN0 3 are ⁇ 32 and ⁇ 120 nm respectively as can be seen from Figures 3C and 3D.
  • the NPs synthesized using higher concentrations of AgN0 3 (1.0-4.0 mmol/L) are found to be of mixed morphology. Triangular, cubical and hexagonal particles are also visible along with the spherical ones as can be seen in Figure 3D.
  • the DLS traces of PAH-Ag NPs synthesized using various concentrations of AgN0 3 are shown in Figure 2C.
  • the synthesized PAH-Ag NPs are centrifuged to remove the unassociated PAH present in the solution and re-dispersed in water before analysis.
  • the average particle size of NPs synthesized using 0.2 - 4.0 mmol/l of AgN0 3 is found to be in the range of 45-140 nm as shown in Table 1 above.
  • the PDI of the NPs are in the range of 0.2-0.4 suggesting reasonably narrow size distribution.
  • the presence of a minor peak in the range of 1-20 nm, possibly due to the formation of un-stabilized Ag NPs in small proportions affected the overall PDI to some extent.
  • the average particle sizes obtained from the DLS studies are somewhat higher than those of the data (25-130 nm) obtained from TEM analysis. This could be attributed to the apparent hydrodynamic volume of the PAH-Ag NPs in solution. Importantly, the TEM and DLS data suggested that, particles with predetermined sizes may be synthesized by controlling the ratio of AgNC ⁇ : PAH in the reaction mixture.
  • AAS analysis also reveals the presence of a substantial amount (91.5-99.7%) of organic constituents in the NPs further confirming the formation of PAH-Ag NPs.
  • the Zeta potential surface charge of the NPs
  • the average particle size is dependent on the pH of the reaction mixture. As can be seen from Figure 7, the average particle dia is found to be
  • NPs is carried out under ambient atmospheric conditions without any additional reagent.
  • anti-tumor drug is loaded into the PAH-Ag NP matrix.
  • both drug Doxorubicin or Dox hereinafter
  • PAH-Ag NPs are encapsulated into a pH responsive hydrogel through in-situ cross-linking.
  • Dox is chosen as the drug in this particular case, as the presence of -CONHNH 2 in PAH provides the opportunity to load the drug both by (i) chemical i.e. through formation of acid labile hydrazone linkage attachment and (ii) physical through hydrogel formation and encapsulation.
  • other drugs such as Taxol, Camptothecin, Paclitaxel, Everolimus etc can also be used.
  • a suitable cross-linker e.g. acrylic acid can be used to prepare the hydrogel at room temperature through known physical cross-linking procedure using acrylic acid.
  • suitable hydrogel forming agents such as dimethyl 2,2'-dithioacetic acid, dimethyl malonate , polyethylene glycol etc can also be used for the purposes of this process which would be known to the person skilled in the art.
  • the yellow colored PAH-Ag NP solution changes to red after addition of Dox.
  • UV-Vis spectroscopic analysis supported the presence of both PAH-Ag NPs and Dox by displaying peaks around 406 and 500 nm respectively as shown in Figure 4B. Gelation occurred after 3 hour of the addition of acrylic acid. The gel is purified by washing with water.
  • Dox is loaded into the PAH-Ag NPs (d aVg «46 nm) to prepare PAH-Ag-Dox NPs.
  • a relatively hydrophobic and planer drug camptothecin (CPT) is a better option for entrapment experiment.
  • CPT camtothecin
  • THF may be used as the medium.
  • the unreacted drug can be removed by dialyzing the loaded NPs in water for 24 hour at room temperature.
  • the hydrophobic drug entrapment (62.5%) and loading efficiencies (6.3%) improves significantly compared to that of the Dox.
  • the release of CPT can be monitored at pH 5.4 found to be in tumor microenvironment. A maximum of ⁇ 78% of the total loading is found to be released over a period of 70 hours as determined from the HPLC analysis as shown is Figure 8.
  • FIG. 4D shows the difference between viability inhibition potential of CPT loaded PAH-Ag NPs and free CPT against MCF-7 cells.
  • Figure 4E is the fluorescence microscopic images of intracellular uptake of PAH-Ag- ITC NPs in MCF-7 cells.
  • PAH-Ag-CPT NPs PAH- Ag NPs (5 Mg/ml) and CPT (0.3 Mg/ml) exhibits lower inhibitory rate (79% and 84% respectively) on MCF-7.
  • the cellular uptake of PAH-Ag NPs has been time dependant and localization of RITC tagged PAH-Ag NPs within the cytosols of MCF-7 cells is visible in the fluorescence microscopic images as illustrated in Figure 4E.
  • PAH-Ag NP has been synthesized by adding AgN0 3 (20 ⁇ , 0.4 mmol/l) to the aqueous solution of PAH (2.0 mL, 30% w/v) at room temperature. To the yellow colored NP solution. Dox (1.0 mg, 1.83 ⁇ ⁇ ) is added and the mixture is stirred for 5 min at room temperature. To the above mixture, acrylic acid (0.4 mg, 5.6 mmol) is added as cross linker and the red colored solution is kept undisturbed at room temperature for 3 hour to prepare the hydrogel.
  • the Dox and PAH-Ag NP encapsulated hydrogel is immersed in a pH 5.0 solution. The temperature of the solution is maintained at 37 °C. After a selected time intervals, the UV-Vis spectroscopic analysis of the media is recorded. The % release was quantified by comparing the absorbance of the media after different time intervals to that of the known initial concentration of Dox.
  • PAH-Ag-CPT NPs Powdered PAH-Ag NP (40 mg) is dispersed in 10 mL of milli-Q water and to it THF solution (2 ml) of CPT (4 mg, 11.5 ⁇ ) is added in drop wise manner with constant stirring. The mixture is stirred at room temperature for 12 hour. The resulting mixture is dialyzed for 4 hour using SMALL WONDER LYZER ⁇ (10- 12 kDA) dialysis bag. The dialyzed CPT encapsulated PAH-Ag NPs are centrifuged at 12500 rpm for 20 min to separate any free CPT. The final CPT loaded PAH-Ag NPs were dried by lyophilization for further studies.
  • Loading efficiency of PAH-Ag-CPT NPs A mixture of PAH-Ag-CPT NPs (1.0 mg) and THF (10 mL) is kept in the incubator at 37 °C for 4 hour to disperse the NPs. The UV- Vis spectroscopic analysis of the dispersion is recorded. The amount of loaded CPT (Wi) is determined from the absorbance using a standard calibration curve. The loading efficiency and entrapment efficiency are then determined.
  • Controlled release of CPT from PAH-Ag-CPT NPs The PAH-Ag-CPT NPs are taken in the dialysis bag and dipped in 10 ml of pH 5.4 buffer solution. The temperature of the medium is maintained at 37 °C with constant stirring. HPLC analysis of the aliquots collected after predetermined time intervals is recorded to determine the extent of release as shown in Figure 9. A mixture of acetonitrile and 0.1% TFA water is used as mobile phase and the flow rate is maintained at 0.8 ml/min for the HPLC studies. The integration of peak at the retention time of 5.3 min is used to quantify the release.
  • Example 8 The integration of peak at the retention time of 5.3 min is used to quantify the release.
  • the modified NP exposed cells are washed with PBS, and then MTT (10 ⁇ , 5 mg/ml) is added into each well.
  • the cells are incubated for an additional 4 hour, followed by removal of media, and addition of 100 ⁇ DMSO into each well to dissolve insoluble formazan formed by the mitochondrial dehydrogenase in live cells.
  • the absorbance of dissolved formazan is measured using a FLUOstar Omega microplate reader (BMG LABTECH) at 530 nm.
  • the cell viability is expressed as the percentage of live cells relative to the control. All experiments are performed in triplicate, and the data are presented as the averaged results with standard deviation.
  • RITC labeled PAH-Ag NPs are prepared by a known method. A solution of RITC in DMSO (1 mg/ml) is added to an aqueous suspension of PAH-Ag NPs (25 mg) at pH 8 in drop wise manner. The resulting mixture is then stirred for 24 hour in the dark. Thereafter, RITC labeled PAH-Ag NPs are dialyzed against DMSO:H 2 0 (10:90, v:v) mixture for 24 hours to remove the unreacted dye. Finally the resulting PAH-Ag-RITC NPs are lyophilized and used for subsequent studies.
  • In-vitro Cellular uptake of fluorescent labeled PAH-Ag NPs For qualitative analysis of cellular uptake, the harvested MCF-7 cells are seeded on 18 mm cover slips in a 12 well culture plate at a density of 5 ⁇ 10 4 cells/well and incubated overnight for cell attachment. Attached cells are incubated with 50 ⁇ g/ml of RITC labeled PAH-Ag NPs over different time periods at 37 °C in C0 2 incubator. Thereafter, cells on cover slips are washed with PBS to remove the free NPs outside of the cells. Washed cells are fixed with chilled methanol at 4 °C for 30 minutes. Nuclei are stained with DAPI and observed under Nikon flstained wi microscope equipped with a camera.
  • PAH-Au NPs are synthesized using a similar procedure as described above in relation to that of the PAH-Ag NPs.
  • Fig. 10 A addition of various concentration of AuCI solution in dioxane to the aqueous solution of 0.02 g/ml PAH resulted in formation of PAH-Au NPs under room temperature conditions within 10 minutes of reaction time.
  • the colour of the solution is found to be white for 0.02mmol/l of AuCI to almost dark brown in case of 4.0 mmol/l of AuCI as the reactant.
  • This reaction is also found to be quantitative.
  • Au° being more stable and non toxic, the presence of any uncapped Au does not lead to toxicity related issue as in the case of Ag. Any excess PAH at this stage can also be removed by centrifuging the reaction mixture.
  • the resulting PAH-Au NPs are separated in similar fashion by dialyzing the reaction mixture in a suitable apparatus.
  • the particle size is again found to be variable on the concentration of AuCI as observed in case of PAH-Ag NPs.
  • the UV-Vis maximum of the PAH-Au NPs changed with the change in size.
  • a bathochromic shift in the m a of the solution from 540 to 560 nm was noticed with increase in the average particle size from 50 to 130 nm.
  • the average particle diameter was in the range of 50 to 130 nm as shown in DLS traces of the samples as shown in Fig. 10 D.
  • PAH-Au NPs are utilized to encapsulate the drug molecules using similar procedure to that followed in case of PAH-Ag NPs. Particularly similar one pot synthesis of drug loaded hydrogel formation reaction is carried out using Dox , PAH- Au NPs and acrylic acid and very similar product characteristics are obtained. Similar pH dependent release data of the drug in tumour micro environment is obtained when PAH-Au NPs are loaded with Camptothecin both in direct and sustained release hydrogel formulation.
  • the color of solution turned to light purple suggesting PAH-Au NP formation.
  • the aqueous dispersion of PAH-Au NP is then dialyzed for 24 hours to remove unreacted starting materials.
  • the solution is then centrifuged to isolate the resulting NPs.
  • the final product is dried by lyophilization prior to characterization.
  • PAH-Ag/Au-drug NPs as synthesized herein have the potential for various therapeutic applications.
  • Stable PAH stabilized Ag/Au NPs with predetermined particle size may be synthesized at room temperature without the need of using any additional reagent, complex reactants and process.
  • Drugs may be conveniently encapsulated in PAH-Ag/Au NPs and delivered in a controlled manner for various biomedical applications.
  • the NPs along with drugs may also be trapped inside an injectable hydrogel for controlled release applications.
  • the whole process of Ag/Au NP synthesis and drug loading can be conducted in a single pot and does not warrant any complicated post purification procedure. Furthermore, the loading and release of the drug may be tailored depending upon the suitability of use.

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Abstract

There is disclosed a process for preparation of polyacryloyl hydrazide stabilized metal nano particles such as PAH stabilized silver or gold nano particles comprising reacting aqueous solution of a homo or co-polymer of acryloyl hydrazide (PAH) with corresponding metal salt solution in ambient conditions. There is further disclosed an one pot process for preparation of drug loaded polyacryloyl hydrazide stabilized metal such as silver or gold nano particles. The invention also discloses such polyacryloyl hydrazide stabilized metal nano particles as well as its drug loaded and sustained release formulation.

Description

A PROCESS FOR THE PREPARATION OF POLYACRYLOYL HYDRAZIDE STABILIZED METAL NANO PARTICLES AND THE PRODUCTS OBTAINED THEREBY
Field of Invention:
This invention relates to an one step process for preparing metal nano particles such as silver or gold nano-particles and in particular to a polyacryloyl hydrazide (PAH) stabilized silver (Ag) or gold (Au) nano particles (PAH-Ag/Au NPs) suitable for biomedical and drug delivery applications. This invention further relates to an one pot synthesis of drug loaded polyacryloyl hydrazide stabilized silver/gold nano particles from the above PAH-Ag/Au NPs and the product obtained thereby as well as to a process of synthesis of a hydrogel based sustained release formulation thereof. The Ag/Au NPs of the invention has controlled and narrow size distribution.
Background of the invention:
Nano particles are of great scientific interest as they are, in effect, a bridge between bulk materials and atomic or molecular structures. The properties of materials change as their size decreases to the nano scale and thereby the percentage of atoms at the surface of the material becoming significantly higher. This reduction in size leads to many interesting and sometimes unexpected properties.
Silver nano particles are nano particles of silver. The nano particles in respect of silver are with a particle size generally between 1 nm to 1000 nm in diameter in average, more specifically between 1-100 nm in diameter in average. Generally a plurality of particle diameters or sizes exist in the nano particles. However great emphasis is given to the narrow range of relatively smaller particle sizes as well the shape of the nano particles for various applications.
Synthesis and surface grafting of silver nanoparticles (Ag NPs) have gathered huge interest in recent times due to their useful antimicrobial properties, catalytic activities in the form of a reducing/oxidizing agent, electrical and thermal conductivity and optical properties. Owing to the above interesting properties, Ag NPs have found applications as sensors, catalysts in redox reactions, electronic devices and antimicrobial agents. The biocidal activities of bare Ag NPs though interesting, is associated with potential toxicity towards cells due to release of Ag+ cations from Ag NPs. Therefore, efforts were made to immobilize Ag NP into a biocompatible material, typically a polymer matrix to resist oxidation (cf. AshaRani, P. V.; Mun, G. L. K.; Hande, M. P.; Valiyaveettil, S. ACS Nano 2009, 3, 279-290. Eby, D. M.; Schaeublin, N. M.; Farrington, K. E.; Hussain, S. M.; Johnson, G. R. ACS Nano 2009, 3, 984-994.).
For various medical applications, polymeric capping agents were preferred over their low molecular weight analogs such as sodium citrate, since the later is known to possess unfavorable anticoagulant activity (cf. Ocwieja, M.; Adamczyk, Z. Langmuir 2013, 29, 3546-3555). The polymer matrix further helped by releasing the Ag NPs in a controlled manner to achieve desired bactericidal effect and avoid eukaryotic toxicity (cf. Zhu, Y.; Morisato, K.; Li, W.; Kanamori, K.; Nakanishi, K. ACS Appl. Mater. Interfaces 2013, 5, 2118-2125 & Liu, J. Y.; Sonshine, D. A.; Shervani, S.; Hurt, R. H. ACS Nano 2010, 4, 6903-6913). However these publication does not disclose any hydrazide functionality but glutathione, cysteine etc.
Another problematic aspect of traditional Ag NP synthetic procedure is the use of inorganic or organic reducing agents. The biocompatibility of the resulting Ag NPs containing trace amount of these toxic impurities is questionable. Therefore, the focus has shifted to the development of benign and green procedures involving polymeric reagents to synthesize the NPs for various biomedical applications. For example, polyamines were used as nanoreactors to synthesize NPs due to their efficient capping ability (cf. Dai, J.; Bruening, M. L. Nano Lett, 2002, 2, 497-501 & Zhou, B.; Zheng, L; Peng, C; Li, D.; Li, J.; Wen, S.; Shen, M.; Zhang, G.; Shi, X. ACS Appl. Mater. Interfaces 2014, 6, 17190-17199).However, in these publications also, an additional agent (NaBH4) or energy source (UV light) was utilized for reduction of metal ions.
Therefore a new synthetic procedure based on polymeric reducing and capping agents would be highly beneficial for future drug delivery applications.
The role of hydrazine as an effective reducing agent for various functional groups is well documented in literature. US patent 8147908 issued to Xerox Corporation discloses the use of hydrazine or substituted hydrazines as one of the reagents used in the synthesis of organoamine stabilized silver nano particles.
Use of hydrazine and/or substituted hydrazine was envisaged because of hydrazines having solubility in water, polar or non-polar organic solvents depending on the substitution; having strong to weak reducing ability depending on the substitution; and nonexistence of non-volatile metal ions as in other reducing agents such as, for example, sodium borohydride, which would facilitate the removal of by-product or unreacted reducing agent.
However, the above publication discloses a four step process containing adding a first amount of organoamine in a solvent followed by addition of silver salt, a second amount of organoamine and an organohydrazine in different weight ratios and in different temperature conditions. Therefore such process becomes much complicated. Further biocompatibility of the different organoamines for medical applications is not confirmed and the silver nano particles produced by the above process is mainly used in the manufacturing of thin film transistors.
Patent publication US 20120225126 discloses a solid state polymer stabilized Ag NP synthesizing process. However, this publication requires a mechanical milling process and the polymers used for the process such as polyvinyl pyrrolidone (PVP) may not be suitable for internal drug delivery applications. Moreover, the publication does not teach any method to overcome the complexity of the reaction process associated with organic polyamines and hydrazides rather it teaches away towards a solid state process to avoid the complexity of a liquid state process for the preparation of Ag NPs.
The use of amines or hydrazides in Ag NP preparation process becomes more complicated because is seen that polyamides are not capable of reducing metal ions while hydrazines are not capable of capping the metal. Therefore, the prior art processes for preparing Ag NPs involving polyamines or hydrazines requires additional agents and complex processes to achieve size controlled metal NPs synthesis. None of the above teachings suggested that metal nano particles particularly silver nano particles can be synthesized in liquid state through a simple one step process using polyamides or hydrazines without involving any solvent and there would be a near quantitative formation of silver nano particle complexes.
Similarly, ligand stabilized Au nano particles are reported to have been prepared from Aum chlorides and some of which can be used in drug delivery complexes. US20140186263 discloses a maleimide-functionalized gold nanoparticle comprising a ligand monolayer, wherein said ligand monolayer includes at least a PEG-thiolated ligand.. HAuCI4 i.e. AuCI3 in acidulated water is capped with polyethylene glycol and thereafter made to react with a furan-protected maleimide-PEG-thiol to produce a furan-protected maleimide-functionalized gold nanoparticle, followed by heating the furan-protected maleimide-functionalized gold nanoparticle intermediate under conditions suitable for removal of the furan-protection group and to provide the maleimide-functionalized gold nanoparticles. Therefore, the preparation process including the reactants disclosed in the specification is quite complex.
On the other hand, Gold1 halides such as AuCI and AuBr are seldom used to prepare gold nanoparticles but because of their non-stable character, a Iky I amines such as oleylamine or octadecylamine can be used for reduce the gold halides to gold nanoparticles c.f. Lu et al; PMC 2008; Chemistry. 2008; 14(5): 1584-1591. However, The AuNPs synthesized through this procedure are bare NPs. These NPs cannot be used to load drug for targeted delivery application. These NPs also lack nanogel formation ability. Further, the use of low molecular weight amine as reducing agents, the resultant gold nano particles are not useful for biomedical applications due to toxicity issues associated oleylamine or octadecylamine.
Thus there is always a need for providing an easy synthetic process for polyamine/polyhydrazide stabilized metal nano particles which can be utilized in biomedical applications including drug delivery agents.
Objects of the invention:
It is the primary object of the invention to synthesize polyamine/polyhydarzide stabilized metal particularly, silver nano particles suitable for biomedical applications; It is another object of the invention to provide a simple and one step process of preparing a polyamine/polyhydrazide stabilized silver nano particles;
It is yet another object of the invention to provide a simple and one pot synthetic process for drug loaded polyamine/polyhydrazide stabilized silver nano particles; It is a further object of the invention is to provide a simple process for preparing a drug loaded polyamine/polyhydrazide stabilized silver nano particles trapped in a hydrogel for slow release formulation.
It is another object of the invention to synthesize polyamine/polyhydarzide stabilized gold nano particles suitable for biomedical applications;
It is yet another object of the invention to provide a simple and one step process of preparing a polyamine/polyhydrazide stabilized gold nano particles;
It is a further another object of the invention to provide a simple and one pot synthetic process for drug loaded polyamine/polyhydrazide stabilized gold nano particles;
It is yet further object of the invention is to provide a simple process for preparing a drug loaded polyamine/polyhydrazide stabilized gold nano particles trapped in a hydrogel for slow release formulation.
Summary of the invention:
Accordingly the present invention provides a process for preparation of polyacryloyi hydrazide (PAH) stabilized metal nano particles comprising reacting aqueous solution of an a cry I oy I hydrazide polymer with a suitable metal salt solution in ambient conditions.
The invention further provides a process for preparation of drug loaded polyacryloyi hydrazide stabilized metal nano particles comprising
(i) reacting an aqueous solution of acryloyl hydrazide polymer with a metal salt solution in ambient conditions to form polyacryloyi hydrazide stabilized metal nano particles; and
(ii) subsequently adding a suitable drug to the aqueous solution of said polyacryloyi hydrazide stabilized metal nano particles under ambient conditions to form drug loaded polyacryloyi hydrazide stabilized metal nano particles. The invention also provides a process for preparation of a sustained release formulation of drug loaded polyacryloyi hydrazide stabilized metal nano particles comprising
(i) reacting an aqueous solution of acryloyi hydrazide polymer with a metal salt solution in ambient conditions to form polyacryloyi hydrazide stabilized metal nano particles; and
(ii) subsequently adding a suitable drug and an aqueous hydrogel forming agent to an aqueous solution of said polyacryloyi hydrazide stabilized metal nano particles under ambient conditions in the same reaction container to form a hydrogel capped drug loaded polyacryloyi hydrazide stabilized metal nano particles.
Said metal nano particles of the invention are acryloyi hydrazide (PAH) homo or copolymer stabilized siver or gold nano particles. Non limiting examples of such polymer are PAH or PAH-co-polyethylene glycolacrylate or PAH-block-polyethylene glycolmethylacrylate (PAH-PEG MA).
Said metal salt is silver nitrate or aurum chloride at a preferred concentration in the range of 0.02 mmol/l to 4 mmoL/l and more preferably 0.2 mmol/l. The concentration of said aqueous solution of PAH is preferably 0.02 g/ml.
Said PAH-Ag nano particles have an average particle diameter in the range of 8-141 nm and preferably between 20- 30 nm. The average particle diameter of the metal nano particles is controllable by controlling the concentration of said metal solution and/or by controlling the pH of the reaction mixture.
The invention further provides a polyacryloyi hydrazide stabilized pH responsive silver or gold nano particle and a polyacryloyi hydrazide stabilized pH responsive silver or gold nano particles.
The invention also provides an antitumor drug loaded polyacryloyi hydrazide stabilized silver or gold nanoparticles and an hydrogel based slow release formulation of drug loaded polyacryloyi hydrazide stabilized silver or gold nanoparticles. Preferably said hydrogel forming agent is selected from acrylic acid, dimethyl 2,2'- dithioacetic acid or dimethyl malonate and preferably the drug is selected from camtothecin, doxorubicin, taxol, paclitaxel, everolimus etc.
The invention and its scope of protection sought for would be clearly understood from the following detailed disclosure of the invention and the accompanying draiongs and as defined in the appended claims.
Brief description of the accompanying drawings:
Figure 1 is a schematic illustration of PAH-Ag NPs formation and drug encapsulation followed by pH dependent controlled release of the drug and silver nano particles according to the invention.
Figure 2A-2C show as below:
A: indicates coloration of the PAH-Ag NPs of the invention under different AgN03 concentration;
B: is a UV-Vis spectra of PAH-Ag NPs formed after diluting the original reaction mixtures by 40 times; and
C: is DLS traces of PAH-Ag NPs synthesized using different concentrations (0.2 - 4.0 mmol/l) of AgN03 and 2 wt% solution of PAH.
Figures 3 A-F show the HRTEM images of PAH-Ag NPs synthesized using
A: 0.2 mmol/l of AgN03;
B: is the particle size distribution for "A";
C: 0.5 mmol/l of AgN03;
D: is the particle size distribution for "C"
E: 2.2 mmol/l of AgN03;
F: is the particle size distribution for "E"
G: 4.0 mmol/l of AgN03, and
H shows the diffraction pattern of a PAH-Ag NP synthesized using 4.0 mmol/l of AgN03 from HRTEM.
Figures 4 A-E show different spectroscopic results, wherein
A is the visual observation of NP formation, drug loading and hydrogel formation stages; B is the UV-Vis spectra of the PAH-Ag NP and drug loaded PAH-Ag NP;
C gradual release of PAH-Ag NP and drug from hydrogel;
D shows the difference between viability inhibition potential of CPT loaded PAH-Ag NPs and free CPT against MCF-7 cells; and
E is the fluorescence microscopic images of intracellular uptake of PAH-Ag-RITC NPs in MCF-7 cells.
Figure 5 shows the FT-IR spectrum of the lyophilized NPs of the invention.
Figure 6 is the absorbance profile of aqueous NPs of the present invention.
Figure 7 represents the average particle size variation of the NPs of the present invention vis-a vis pH of the reaction mixture, which is a characteristic behavior of pH responsive nanogel.
Figure 8 is the release profile of CPT at pH 5.4 from PAH-Ag-CPT NPs of the present invention.
Figure 9A-9C show: (A) Color and (B) UV-Vis traces and (C) DLS traces of PAH-PEGMA capped Ag NPs synthesized using various concentration of AgN03
Figure lOA-lOC show: (A) Color and (B) UV-Vis traces of the PAH-Au NPs synthesized using various concentration of AuCI and (C) Absorption maximum versus [AuCI] plot of PAH-Au NPs of varying particle size.
Figure 10D shows DLS traces of PAH-Au NPS synthesized using varying concentration of AuCI.
Detailed description of invention:
With a view to remedy the drawbacks of the prior art and to synthesize suitable polymer stabilized metal NPs such as AgNPs and AuNPs through a very simple procedure, which are useful for biomedical drug delivery application, an extensive study of the different properties of the various polymers of amines and hydrazides including homo and copolymers of acryloyl hydrazides has been undertaken. On such study, it is now found that the abovementioned objects of the invention can be accomplished by using homo/co polymers of acryloyl hydrazide for stabilizing Ag/Au
NPs. Example of such polymers includes but is not limited to polyacryloyl hydrazide
(PAH), PAH-block-polyethylene glycolmethylacrylate (PAH-PEGMA) etc. These polymers can act both as reducing and stabilizing or capping agent in the process of preparing metal nano particles at the same time these PAH based homo /co polymers are very effective in achieving size controlled metal NPs synthesis particularly Ag/Au NP synthesis and subsequently such NPs can be used in drug delivery applications.
Polymers of acryloyl hydrazide can be represented by a general structure of formula 0)
Figure imgf000010_0001
0)
Wherein, Ri and R2 are H or CH3, in a preferred embodiment Ri is CH3 and R2 is H; R3 is H or a linear or branched CpH2p+i (p is a natural number), in a preferred embodiment R3 is H;
R4 may be an oligo or polymeric polyethylene glycol moiety, having a Molecular Weight of 300-5000, typically 500.
n can be 0 or a +ve integer and m can be any number in between 10-10000.
Exemplary structures of the polymers can have a molecular structure of formula (ii) and (iii);
Figure imgf000010_0002
(ii) (iii) Use of PAHs for the synthesis of metal NPs is found to be multifold. Polyacryloyl hydrazide homo or copolymers possess hydrazide functionality in each repeating unit. The presence of large amount of carbazide groups makes PAHs a stronger reducing agent for the metal ion precursors in comparison to that of normal polyamines. The -NH2 group present in the pendant hydrazide moiety can swiftly cap the surface and stabilize the resulting NPs. Presence of hydrophilic PAH on the surface of metal NPs would assist easy dispersibility in aqueous media. More importantly, presence of PAH (pH«8.7) on the surface of Ag NPs resists the oxidation of Ag° to Ag+ and thereby reduce the toxicity associated with Ag NPs. The pH responsiveness of PAH can be swiftly utilized to encapsulate various drug molecules in the NPs and to release them in a pH controlled manner under physiological conditions. PAH also forms cytocompatible, injectable and stimuli responsive hydrogels with a range of crosslinkers. Therefore, the procedure may be extended to encapsulate the synthesized Ag/Au NPs along with drug molecules in-situ through formation of hydrogels.
Experimentally, It is now found that homo or copolymer of acryloyl hydrazide such as PAH or PAH-co-polyethylene glycolacrylate is highly suitable for an one step reagent free synthesis of stable and non-toxic Ag/Au NPs. Further it has now been found that PAH-Ag/Au NPs formed by the process of the invention has controlled and narrow range of particle size as well as it can be formed with a predominantly predetermined shape which are important aspects of preparing the biomedical drug delivery complexes.
For these purposes in an exemplary embodiment, PAH with Number Average Molecular Weight( M„) = 62000 and Polydispersity Index (PDI )= 1.6 has been synthesized and purified in a known manner and in a reaction container aqueous solution of such PAH is made to react with aqueous AgN03 at room temperature to prepare PAH stabilized Ag NPs
Figure 1 is a schematic illustration summarizing the whole process as would be described below. As shown therein, the inventive PAH-Ag NPs has the added advantage of encapsulating and releasing drugs in a controlled manner. PAH is soluble is water, but a polar solvent such as DMF, NMP etc may also be used. Preferably silver nitrate is used as the silver salt but other salts such as silver halides, sulfates, acetates may also be used for the above reaction.
In an exemplary embodiment the Ag NPs are synthesized by adding different concentrations of aqueous AgN03 to 0.02 gm/ml aqueous solution of PAH (pH«8.7) at room temperature. The hydrazide functionality present in PAH served as both reducing agent for AgN03 and surface capping agent of the resulting NPs. The formation of Ag NPs has been swift and the reaction is completed in about 5-20 minutes under ambient conditions and a characteristic yellow color (cf. Figure 2A) of the reaction mixture appears within around 15 minutes of the addition of 0.2 mmol/l AgN03. Even lesser concentration of AgN03/ i.e. upto 0.02 mmol/l is quite effective in producing PAH stabilized silver nano particles with desired characteristics. However, for ease of reaction 0.2 mmol/l of AgN03 is the most desirable concentration. This is schematically shown in Figure 1 wherein (i) shows the _CONHNH2 moiety and PAH- AgNPs are shown in (ii).
Slight heating of the reaction mixture i.e. up to 100° c may be preferred for enhanced reaction kinetic but is not essential. Most importantly the reaction is found to be quantitative. The resulting PAH-AgNPs can be purified by diaiyzing the reaction product thus formed.
The pH of the reaction mixture varies in between 8.3 to 8.9 depending upon the concentration of PAH. Typically pH=8.4 solution is used to synthesize the metal NPs. PAH being a base controls the pH of the aqueous solution.
As it is the aim to provide a one step process without the need to purify the resulting PAH-Ag NPs, it is important that AgN03 solution be added in a near quantitative manner. PAH may be used in excess rather than AgN03 so that toxicity associated with Ag+ ion can be avoided. Any unreacted PAH may be removed in this stage by way diaiyzing and/or centrifuging the reaction mixture. The reaction being quantitative carefully executed one completely eliminates the possibility of any uncapped metal NPs and the need for separation of resulting mixture becomes minimal. PAH is attached with the surface of the silver nano particles and is considerably stable. The silver nano particles and PAH form the complex through chemical bond and/or other physical attachment such as covalent bonding, hydrogen bonding, coordination complex bonding, ionic bonding or a mixture of different chemical bonds, while the physical attachment may be through van der Waals' forces, dipole- dipole interaction or a mixture of different physical attachments.
As shown in figure 2B, the UV-Vis spectrum of the above reaction mixture displayed a
~ 406 nm, characteristics of the polymer stabilized Ag NPs evidencing formation of PAH-Ag NPs. Color of the reaction mixture gradually changes from yellow to brown upon increasing the concentration of AgN03 in the solution suggesting a possible change in the particle size. The surface plasmon band is typically observed in the range of 406-425 nm for samples synthesized using various concentrations (0.2-4.0 mmol/l) of AgN03 and the absorbance gradually increased with increased concentration of AgN03.
Table 1 below shows the particle size, yield and surface charge of Ag NPs produced by the inventive process in different AgNC>3 concentrations , wherein *the FWHM is determined from UV-Vis spectroscopic analysis, "the wt% of Ag in PAH-Ag NP is determined from AAS analysis, *the average particle diameter (davg), PDI and Zeta potential (ς) are determined from DLS analysis.
[AgN03] davg Ag (wt%)# PDI1 FWHM
(mmol/L) (nm)1 (mV) (nm)*
0.2 45.7 0.3 □6.9 0.4 42
0.5 50.4 2.5 □6.3 0.4 60
1.0 75.0 3.1 □2.4 0.4 62
1.4 100.1 5.9 □4.5 0.3 67
2.2 105.9 6.2 □4.9 0.2 68
4.0 140.9 8.5 □ 1.2 0.3 83
Table 1 It can be seen from the above table that a relatively narrow full width half maximum (FWHM) » 42-68 nm is obtained for the samples synthesized using 0.2 - 2.2 mmol/l of AgN03 indicating the particle size distribution is reasonably controlled . The particle size, morphology and uniformity of size distribution are determined by HRTEM, DLS and FESEM analysis.
As shown in Figure 3A, the Ag NPs synthesized using 0.2 mmol/l of AgN03 are mostly spherical and the particle diameters are in the range of 8-35 nm.
Form Figure 3B, it can be seen that the size distribution is considerably narrow, with the particles possessing diameters in the range of 20 - 30 nm are the most abundant ones.
FESEM images also displayed clusters of NPs possessing size distribution similar to that of the HRTEM data. It is seen that the average size of NPs increases with the increase in the amount of AgN03 in the reaction mixture. The average size of the PAH-Ag NPs synthesized using 0.5 and 2.2 mmol/l of AgN03 are ~32 and ~120 nm respectively as can be seen from Figures 3C and 3D. Interestingly, the NPs synthesized using higher concentrations of AgN03 (1.0-4.0 mmol/L) are found to be of mixed morphology. Triangular, cubical and hexagonal particles are also visible along with the spherical ones as can be seen in Figure 3D. From the above data it can be inferred that it is possible to control the shape of the resulting Ag NPs using selective concentration or molecular weight of PAH. However it is also seen that the control over polydispersity is compromised to some extent, when the NPs are synthesized using very high concentration (4.0 mmol/l) of AgN03 as shown in Figure 3G. It can be further seen that the sizes of the resulting PAH-Ag NPs are distributed over a range of 40-200 nm. Figure 3H showing the electron diffraction pattern confirms packing of crystalline Ag NPs into a face centered cubic lattice with a typical lattice fringe of 2.2 A*.
The DLS traces of PAH-Ag NPs synthesized using various concentrations of AgN03 are shown in Figure 2C. The synthesized PAH-Ag NPs are centrifuged to remove the unassociated PAH present in the solution and re-dispersed in water before analysis.
The average particle size of NPs synthesized using 0.2 - 4.0 mmol/l of AgN03 is found to be in the range of 45-140 nm as shown in Table 1 above. The PDI of the NPs are in the range of 0.2-0.4 suggesting reasonably narrow size distribution. In some compositions, the presence of a minor peak in the range of 1-20 nm, possibly due to the formation of un-stabilized Ag NPs in small proportions affected the overall PDI to some extent.
The average particle sizes obtained from the DLS studies are somewhat higher than those of the data (25-130 nm) obtained from TEM analysis. This could be attributed to the apparent hydrodynamic volume of the PAH-Ag NPs in solution. Importantly, the TEM and DLS data suggested that, particles with predetermined sizes may be synthesized by controlling the ratio of AgNC^: PAH in the reaction mixture.
FT-IR spectrum of the lyophilized NPs displayed characteristic bands accountable to - C=Ostr and -C-Nstr supporting the surface capping of Ag NPs by PAH chains as seen in Figure 5. AAS analysis also reveals the presence of a substantial amount (91.5-99.7%) of organic constituents in the NPs further confirming the formation of PAH-Ag NPs. The Zeta potential (surface charge of the NPs) is found to be in the range of -22 to -30 mV suggesting moderate stability of the PAH-Ag NPs. The NPs in aqueous solution retained more than 90% of the original absorbance [XmM = 410 nm) till 190 hour as shown in Figure 6 supports the above.
It is also observed that the average particle size is dependent on the pH of the reaction mixture. As can be seen from Figure 7, the average particle dia is found to be
66nm when the reaction mixture is maintained at a pH range of 8.3-8.9.
In the next step, an one pot synthesis of drug loaded PAH-Ag NPs, from the PAH-Ag
NPs is carried out under ambient atmospheric conditions without any additional reagent.
In the exemplary embodiment anti-tumor drug is loaded into the PAH-Ag NP matrix.
In the first approach, both drug (Doxorubicin or Dox hereinafter) and PAH-Ag NPs are encapsulated into a pH responsive hydrogel through in-situ cross-linking. Dox is chosen as the drug in this particular case, as the presence of -CONHNH2 in PAH provides the opportunity to load the drug both by (i) chemical i.e. through formation of acid labile hydrazone linkage attachment and (ii) physical through hydrogel formation and encapsulation. It would be appreciated that other drugs such as Taxol, Camptothecin, Paclitaxel, Everolimus etc can also be used.
In this particular non limiting embodiment, the physical encapsulation and release of Dox along with PAH-Ag NPs has been demonstrated.
A suitable cross-linker e.g. acrylic acid can be used to prepare the hydrogel at room temperature through known physical cross-linking procedure using acrylic acid. However, other suitable hydrogel forming agents such as dimethyl 2,2'-dithioacetic acid, dimethyl malonate , polyethylene glycol etc can also be used for the purposes of this process which would be known to the person skilled in the art. As shown in Figure 4A, the yellow colored PAH-Ag NP solution changes to red after addition of Dox. UV-Vis spectroscopic analysis supported the presence of both PAH-Ag NPs and Dox by displaying peaks around 406 and 500 nm respectively as shown in Figure 4B. Gelation occurred after 3 hour of the addition of acrylic acid. The gel is purified by washing with water. As shown in Figure 4C, the release has been monitored at pH 5.0 after regular time intervals. The absorbance of peaks at 406 nm accountable to Ag NP and the set of peaks around 500 nm accountable to Dox , as shown in Figure 4C, increases with time suggesting release of both NP and Dox from hydrogel matrix . It can also be seen from Figure 4A that quantitative release of Dox occurs over a period of 16 hour as the hydrogel became liquid like due to substantial dissociation of physical cross-linking. The formation of Dox and NP loaded hydrogel is shown in (iii) of Figure 1.
In another embodiment, Dox is loaded into the PAH-Ag NPs (daVg«46 nm) to prepare PAH-Ag-Dox NPs. However, owing to poor loading efficiency (~2.3%) of Dox in the PAH-Ag NPs, a relatively hydrophobic and planer drug camptothecin (CPT) is a better option for entrapment experiment. It may be appreciated that such process of entrapment of the active drug molecule is not restricted to camtothecin (CPT) only and a person skilled in the art will be able to select suitable drugs for such kind of entrapment e.g. Taxol ,Paclitaxel, Everolimus etc. For CPT, THF may be used as the medium. The unreacted drug can be removed by dialyzing the loaded NPs in water for 24 hour at room temperature. Interestingly, with CPT, the hydrophobic drug entrapment (62.5%) and loading efficiencies (6.3%) improves significantly compared to that of the Dox. The release of CPT can be monitored at pH 5.4 found to be in tumor microenvironment. A maximum of ~78% of the total loading is found to be released over a period of 70 hours as determined from the HPLC analysis as shown is Figure 8.
The controlled release character of drug loaded AgNPs both in hydrogel form and normal form are shown in (iv) and (vi) of Figure 1.
In order to assess the efficacy of PAH-Ag-CPT NPs, anti-proliferative effect of these NPs on MCF-7 breast adeno-carcinoma cells has been evaluated using MTT assay. Moreover, the inhibitory concentration (IC50) by constructing a dose-response curve of CPT loaded Ag-nanogels on MCF-7 cells has also been evaluated . Figure 4D shows the difference between viability inhibition potential of CPT loaded PAH-Ag NPs and free CPT against MCF-7 cells. The vertical bars represent the mean ± SD, n = 3 (number of experiments per concentration) **P < 0.01 and ***P < 0.001 and a p- value of <0.05 is considered statistically significant. Figure 4E is the fluorescence microscopic images of intracellular uptake of PAH-Ag- ITC NPs in MCF-7 cells.
From the result above, it is observed that PAH-Ag-CPT NPs with a CPT concentration = 0.06-0.6 Mg/ml significantly inhibits the proliferation of MCF-7 breast adenocarcinoma cells (84-15%) in a dose-dependent manner (IC5o = 0.3 Mg/I).
Significantly it has also been observed that in contrast to the PAH-Ag-CPT NPs, PAH- Ag NPs (5 Mg/ml) and CPT (0.3 Mg/ml) exhibits lower inhibitory rate (79% and 84% respectively) on MCF-7. The cellular uptake of PAH-Ag NPs has been time dependant and localization of RITC tagged PAH-Ag NPs within the cytosols of MCF-7 cells is visible in the fluorescence microscopic images as illustrated in Figure 4E. These results indicate a synergistic effect between the PAH-Ag NP carrier and the drug. Example 1:
Synthesis of PAH-Ag NPs: To an aqueous solution of PAH (2 ml_, 2% w/v), AgN03 solution (20 0.2 mmol/l) is added at room temperature and the mixture is stirred for 30 min. The color of solution turned to light yellow suggesting PAH-Ag NP formation. The aqueous dispersion of PAH-Ag NP is then dialyzed for 24 hours to remove unreacted starting materials. The solution is then centrifuged to isolate the resulting NPs. The final product is dried by lyophilization prior to characterization. Example 2:
Synthesis of PAH-block-PEGMA-Ag NPs: To an aqueous solution of PAH-block-PEGMA (1 ml, 2% w/v), AgN03 solution (20 pL, 0.4 mmol/l) is added at room temperature and the mixture is stirred for 30 min. As shown in Fig. 9A, the color of solution turned to light yellow suggesting PAH-block-PEGMA-Ag NP formation. The aqueous dispersion of PAH-block-PEGMA-Ag NP is then dialyzed for 24 hours to remove unreacted starting materials. The solution is then centrifuged to isolate the resulting NPs. The final product is dried by lyophilization prior to characterization. As shown in Figs. 9B and 9C show the results of the various spectral analysis results characteristic of PAH - block-PEGMA-Ag NPs prepared in this working example.
Example 3:
Preparation of Dox and PAH-Ag NP encapsulated hydrogel: PAH-Ag NP has been synthesized by adding AgN03 (20 μί, 0.4 mmol/l) to the aqueous solution of PAH (2.0 mL, 30% w/v) at room temperature. To the yellow colored NP solution. Dox (1.0 mg, 1.83 μ ιοΙ) is added and the mixture is stirred for 5 min at room temperature. To the above mixture, acrylic acid (0.4 mg, 5.6 mmol) is added as cross linker and the red colored solution is kept undisturbed at room temperature for 3 hour to prepare the hydrogel.
Example 4:
Release of Dox and PAH-Ag NP from pH responsive hydrogel: The Dox and PAH-Ag NP encapsulated hydrogel is immersed in a pH 5.0 solution. The temperature of the solution is maintained at 37 °C. After a selected time intervals, the UV-Vis spectroscopic analysis of the media is recorded. The % release was quantified by comparing the absorbance of the media after different time intervals to that of the known initial concentration of Dox.
Example 5:
Synthesis of PAH-Ag-CPT NPs: Powdered PAH-Ag NP (40 mg) is dispersed in 10 mL of milli-Q water and to it THF solution (2 ml) of CPT (4 mg, 11.5 μπιοΙ) is added in drop wise manner with constant stirring. The mixture is stirred at room temperature for 12 hour. The resulting mixture is dialyzed for 4 hour using SMALL WONDER LYZER · (10- 12 kDA) dialysis bag. The dialyzed CPT encapsulated PAH-Ag NPs are centrifuged at 12500 rpm for 20 min to separate any free CPT. The final CPT loaded PAH-Ag NPs were dried by lyophilization for further studies.
Example 6:
Loading efficiency of PAH-Ag-CPT NPs: A mixture of PAH-Ag-CPT NPs (1.0 mg) and THF (10 mL) is kept in the incubator at 37 °C for 4 hour to disperse the NPs. The UV- Vis spectroscopic analysis of the dispersion is recorded. The amount of loaded CPT (Wi) is determined from the absorbance using a standard calibration curve. The loading efficiency and entrapment efficiency are then determined.
Example 7:
Controlled release of CPT from PAH-Ag-CPT NPs: The PAH-Ag-CPT NPs are taken in the dialysis bag and dipped in 10 ml of pH 5.4 buffer solution. The temperature of the medium is maintained at 37 °C with constant stirring. HPLC analysis of the aliquots collected after predetermined time intervals is recorded to determine the extent of release as shown in Figure 9. A mixture of acetonitrile and 0.1% TFA water is used as mobile phase and the flow rate is maintained at 0.8 ml/min for the HPLC studies. The integration of peak at the retention time of 5.3 min is used to quantify the release. Example 8:
Cell viability assay: Anti-cancer property of the PAH-Ag-CPT NPs against MCF-7 breast adeno-carcinoma cells is determined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltertra-zolium bromide (MTT) assay. Cells are plated at 70-80% confluence and incubated at 37 °C under a humidified atmosphere with C02 (5%) for 24 hour. For treatment exposure, the cells are incubated with PAH-Ag NPs (t 10 μg/ml); CPT (6.3% of 1- 10 μg/ml) or PAH-Ag-CPT NPs (1- 10 μg/ml). After incubation for 24 and 48 hour, the modified NP exposed cells are washed with PBS, and then MTT (10 μΙ, 5 mg/ml) is added into each well. The cells are incubated for an additional 4 hour, followed by removal of media, and addition of 100 μΙ DMSO into each well to dissolve insoluble formazan formed by the mitochondrial dehydrogenase in live cells. After 30 minutes of gentle shaking, the absorbance of dissolved formazan is measured using a FLUOstar Omega microplate reader (BMG LABTECH) at 530 nm. The cell viability is expressed as the percentage of live cells relative to the control. All experiments are performed in triplicate, and the data are presented as the averaged results with standard deviation.
Statistical Analysis: All data are presented as the mean ± standard deviation (SD), and a p-value of <0.05 was considered statistically significant. The difference between three or more groups was analyzed by one-way ANOVA multiple comparisons.
Example 9:
Fluorescent labeling of PAH-Ag NPs with Rhodamine B Isothiocyanate (RITC): RITC labeled PAH-Ag NPs are prepared by a known method. A solution of RITC in DMSO (1 mg/ml) is added to an aqueous suspension of PAH-Ag NPs (25 mg) at pH 8 in drop wise manner. The resulting mixture is then stirred for 24 hour in the dark. Thereafter, RITC labeled PAH-Ag NPs are dialyzed against DMSO:H20 (10:90, v:v) mixture for 24 hours to remove the unreacted dye. Finally the resulting PAH-Ag-RITC NPs are lyophilized and used for subsequent studies.
Example 10:
In-vitro Cellular uptake of fluorescent labeled PAH-Ag NPs: For qualitative analysis of cellular uptake, the harvested MCF-7 cells are seeded on 18 mm cover slips in a 12 well culture plate at a density of 5 χ 104 cells/well and incubated overnight for cell attachment. Attached cells are incubated with 50 μg/ml of RITC labeled PAH-Ag NPs over different time periods at 37 °C in C02 incubator. Thereafter, cells on cover slips are washed with PBS to remove the free NPs outside of the cells. Washed cells are fixed with chilled methanol at 4 °C for 30 minutes. Nuclei are stained with DAPI and observed under Nikon flstained wi microscope equipped with a camera.
In a further exemplary embodiment a similar process of preparation of AuNPs is demonstrated.
PAH-Au NPs are synthesized using a similar procedure as described above in relation to that of the PAH-Ag NPs. As shown in Fig. 10 A, addition of various concentration of AuCI solution in dioxane to the aqueous solution of 0.02 g/ml PAH resulted in formation of PAH-Au NPs under room temperature conditions within 10 minutes of reaction time. The colour of the solution is found to be white for 0.02mmol/l of AuCI to almost dark brown in case of 4.0 mmol/l of AuCI as the reactant. This reaction is also found to be quantitative. However, Au° being more stable and non toxic, the presence of any uncapped Au does not lead to toxicity related issue as in the case of Ag. Any excess PAH at this stage can also be removed by centrifuging the reaction mixture.
The resulting PAH-Au NPs are separated in similar fashion by dialyzing the reaction mixture in a suitable apparatus.
The particle size is again found to be variable on the concentration of AuCI as observed in case of PAH-Ag NPs. As shown in Figs. 10 B and 10 C, the UV-Vis maximum of the PAH-Au NPs changed with the change in size. A bathochromic shift in the ma of the solution from 540 to 560 nm was noticed with increase in the average particle size from 50 to 130 nm.
The average particle diameter was in the range of 50 to 130 nm as shown in DLS traces of the samples as shown in Fig. 10 D.
Presence of PAH on the surface of NPs render these metal nanoparticles necessary pH responsiveness. The PAH-Au NPs are utilized to encapsulate the drug molecules using similar procedure to that followed in case of PAH-Ag NPs. Particularly similar one pot synthesis of drug loaded hydrogel formation reaction is carried out using Dox , PAH- Au NPs and acrylic acid and very similar product characteristics are obtained. Similar pH dependent release data of the drug in tumour micro environment is obtained when PAH-Au NPs are loaded with Camptothecin both in direct and sustained release hydrogel formulation.
Example 11:
Synthesis of PAH-Au NPs: To an aqueous solution of PAH (2 mL, 2% w/v), AuCI solution (20 μί, 0.2 mmol/l) is added at 10 °C and the mixture is stirred for 10 min.
The color of solution turned to light purple suggesting PAH-Au NP formation. The aqueous dispersion of PAH-Au NP is then dialyzed for 24 hours to remove unreacted starting materials. The solution is then centrifuged to isolate the resulting NPs. The final product is dried by lyophilization prior to characterization.
The above results and observations confirm that PAH-Ag/Au-drug NPs as synthesized herein have the potential for various therapeutic applications. Stable PAH stabilized Ag/Au NPs with predetermined particle size may be synthesized at room temperature without the need of using any additional reagent, complex reactants and process. Drugs may be conveniently encapsulated in PAH-Ag/Au NPs and delivered in a controlled manner for various biomedical applications. The NPs along with drugs may also be trapped inside an injectable hydrogel for controlled release applications. The whole process of Ag/Au NP synthesis and drug loading can be conducted in a single pot and does not warrant any complicated post purification procedure. Furthermore, the loading and release of the drug may be tailored depending upon the suitability of use.
Though, the present invention has been described with the help of an exemplary embodiment of polyacryloyl hydrazide homo or copolymer stabilized metal nanoparticles and particularly PAH stabilized silver/gold nano particles it may be appreciated that the scope of the invention cannot be restricted to such exemplary embodiments and various further embodiments may be possible without departing from the scope of the invention as would be evident to a person skilled in the art.

Claims

Claims,
1. A process for preparation of polyacryloyl hydrazide (PAH) stabilized metal nano particles comprising reacting aqueous solution of an acryloyi hydrazide polymer with a suitable metal salt solution in ambient conditions.
2. A process for preparation of drug loaded polyacryloyl hydrazide stabilized metal nano particles comprising
(i) reacting an aqueous solution of acryloyi hydrazide polymer with a metal salt solution in ambient conditions to form polyacryloyl hydrazide stabilized metal nano particles; and
(ii) subsequently adding a suitable drug to the aqueous solution of said polyacryloyl hydrazide stabilized metal nano particles under ambient conditions to form drug loaded polyacryloyl hydrazide stabilized metal nano particles.
3. A process for preparation of a sustained release formulation of drug loaded polyacryloyl hydrazide stabilized metal nano particles comprising
(i) reacting an aqueous solution of acryloyi hydrazide polymer with a metal salt solution in ambient conditions to form polyacryloyl hydrazide stabilized metal nano particles; and
(ii) subsequently adding a suitable drug and an aqueous hydrogel forming agent to an aqueous solution of said polyacryloyl hydrazide stabilized metal nano particles under ambient conditions to form a hydrogel capped drug loaded polyacryloyl hydrazide stabilized metal nano particles.
4. The process as claimed in any of claims 1 to 3, wherein said metal nano particles are polyacryloyl hydrazide (PAH) stabilized silver or gold nano particles
5. The process as claimed in any one of claims 1 to 3, wherein said polymer is a homo or copolymer of acryloyi hydrazide such as PAH, PAH-co-polyethylene glycolacrylate or PAH-block-polyethylene glycolmethylacrylate (PAH-PEGMA).
6. The process as claimed in any on of claims 1-3 , wherein the said metal salt is silver nitrate or aurum chloride at a concentration in the range of 0.02 mmol/l to 4 mmoL/l.
7. The process as claimed in claim 6, wherein the concentration of silver nitrate or aurum chloride is 0.2 mmol/l.
8. The process as claimed in any one of claims 1 to 3, wherein the concentration of said aqueous solution of PAH is 0.02 g/ml.
9. The process as claimed in claim 4, wherein the said PAH-Ag nano particles have an average particle diameter in the range of 8-141 nm.
10. The process as claimed in claim 9, wherein the said Ag NPs have an average particle diameter between 20- 30 nm.
11. The process as claimed in any preceding claim, wherein the average particle diameter of the metal nano particles is controllable by controlling the concentration of said metal solution and/or by controlling the pH of the reaction mixture.
12. The process as claimed in claim 3, wherein said hydrogel forming agent is selected from acrylic acid, dimethyl 2,2'-dithioacetic acid or dimethyl malonate.
13. The process as claimed in claim 2 or 3, wherein the said drug is an antitumor drug.
14. The process as claimed in claim 12, wherein the said drug is selected from camtothecin, doxorubicin, taxol, paclitaxel, everolimus.
15. A polyacryloyl hydrazide stabilized pH responsive silver or gold nano particle.
16. An antitumor drug loaded polyacryloyl hydrazide stabilized silver or gold nanoparticles.
17. A hydrogel based slow release formulation of drug loaded polyacryloyl hydrazide stabilized silver or gold nanoparticles.
PCT/IN2015/000383 2015-02-10 2015-10-12 A process for the preparation of polyacryloyl hydrazide stabilized metal nano particles and the products obtained thereby WO2016128988A1 (en)

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