Novel Zein Based Nanocarriers for Drug Delivery Applications

Nanoparticles prepared from a variety of natural and synthetic polymers have been extensively investigated for drug delivery applications. Compared to synthetic polymers, natural polymers such as protein based polymers have the advantage of ease of availability and are renewable. Most of the proteins that have been used for preparation of nanoparticles are water soluble with limited sustained release characteristics. In contrast water insoluble plant proteins such as zein have the potential for developing controlled release drug delivery systems. However it is challenging to prepare nanoparticles using natural polymers, since multiple variables such as purity, protein composition, molecular weight, isoelectric point, solvent composition and pH influence the preparation of zein nanoparticles. Hence it is important to identify formulation parameters that are required for reproducible nanoparticle product characteristics. Further it is important to .ensure that the protein nanoparticles are biodegradable and non immunogenic. To this end, one of the main objectives was to identify optimal conditions and identify the optimal size for preparation of nanoparticles with regard to drug delivery and biocompatibility. The knowledge derived from these studies was used to design zein based nanocarriers for drug delivery applications. The first objective was to optimize the preparation of zein nanoparticles using pH controlled nanoprecipitation method to produce nananoparticles below 500 nm with a narrow size distribution. The second objective was to study the influence of particle size on blood compatibility, polymorphonuclear uptake (PMN), macrophage uptake, and in vivo immunogenicity of zein nanoparticles. The third objective was to investigate the application of zein nanoparticles for delivery of a model anticancer drug doxorubicin hydrochloride. The fourth objective was to study the feasibility of preparing nanomicelles using polyethylene glycol (PEG) conjugated to zein. The fifth objective was to investigate the application of PEG-Zein micelles for delivery of a model hydrophobic anticancer drug doxorubicin. It was found that maintaining the pH of the zein solution near the isoelectric point is the major factor that controls the particle size of zein nanoparticles. Combination of lecithin and pluronic F68 as stabilizers resulted in smaller particle size in comparison to use of either of them alone. Low concentration of zein and salt concentration resulted in smaller sized nanoparticles. Citrate buffer was found to be a critical factor in preventing particle growth during the freeze drying process. Coumarin (hydrophobic) and rhodamine (hydrophilic) were used as model compounds to study the characteristics of nanoparticles. The optimized parameters resulted in coumarin loaded zein nanoparticles of 256±30nm, with a polydispersity index of 0.42±0.08 and encapsulation efficiency of 78±7 %. In-vitro release of coumarin in pH 7.4 is sustained from zein nanoparticles up to 9 days. There was a significant burst release within the first 24 hours followed by sustained release of coumarin. Remaining 30% of the encapsulated coumarin was released in 9 days. Rhodamine loaded non cross linked and genipin cross linked zein nanoparticles had particle size of 327±18, and 279±20 nm, and encapsulation efficiency of 83±9 % and 67±8 % respectively. In vitro release of rhodamine from non cross linked zein nanoparticles was rapid under acidic pH (pH 1.4) and was further expedited in presence of pepsin. On the other hand in pH 7.4, the release of rhodamine was relatively slower with initial 55 % burst followed by sustained release for about 8 days. However in presence of trypsin (pH 7.4 ), about 90 % ofrhodamine was released within 8 hrs. Zein nanoparticles were cross linked using glutaraldehyde, d,1-sulphur camphoronic acid and genipin. Cross linking was done to reduce burst release, and improve the enzymatic stability of zein nanoparticles. Among three cross linking agents, genipin was identified as optimal cross linking agent for zein nanoparticles. Cross linking of zein nanoparticles improved the sustained release characteristics and enzymatic stability of zein nanoparticles. Zein nanoparticles were non hemolytic irrespective of particle size, and were compatible with blood components. In comparison to positive control (zymosan), zein particles \u3e400nm showed higher phagocytic uptake in polymorphonuclear (PMN) cells, while the uptake was minimal with smaller nanoparticles (\u3c 400nm). Similar uptake of zein nanoparticles was observed in murine alveolar peritoneal macrophages (1774 cells). However the uptake was higher in case of lgG opsonized particles. Anti-zein IgG antibodies, and titers subsequent to primary and secondary immunization of zein nanoparticles in mice were in the increasing order of 452± 16 nm \u3c 7 48±60 nm\u3c coarse suspension. On the other hand smaller zein nanoparticles (143±7) nm did not produce any anti zein antibodies. The results showed that zein nanoparticles \u3c 400 nm are suitable for in vivo applications. The mean diameter, polydispersity index, and zeta potential of doxorubicin loaded zein nanoparticles were 171±8 nm, 0.31±0.02, and-16±3 mVrespectively. It was found that genipin cross linked zein nanoparticles reduced the burst release and improved the stability of particles in acidic pH and enzymes. Hence genipin cross linking was used in the preparation of doxorubicin loaded zein nanoparticles. The encapsulation efficiency of doxorubicin in non cross linked and cross linked zein nanoparticles was 80± 13 % and 74±3 % respectively. DSC studies indicated that doxorubicin was encapsulated inside zein nanoparticles. In vitro release kinetics of doxorubicin from non cross linked particles in saline followed first order kinetics. In citrate buffer pH 7.4, doxorubicin release followed Korsmeyer-Peppas model indicating that the release is controlled by more than one process including diffusion, dissolution and erosion. On the other hand doxorubicin release from non cross linked particles followed Hixon- Crowell model in citrate buffer (pH 7.4), describing that the release is governed by change in surface area and diameter of nanoparticles. Release of doxorubicin from cross linked zein nanoparticles followed Korsmeyer-Peppas (n value less than 0.47) model indicating diffusion controlled release. Doxorubicin loaded zein nanoparticles resulted in 2 to 4 fold reduction of IC 50 values in resistant human cancer cell lines. There was enhanced cell uptake and prolonged retention of doxorubicin from zein nanoparticles. Both cotreatment and pretreatment of blank zein nanoparticles enhanced the cell uptake of doxorubicin in drug resistant cell lines. Further blank zein nanoparticles exhibited Pgp inhibition activity. Doxorubicin loaded zein nanoparticles have increased biodistribution of doxorubicin to tumors, and significantly lower amount in other organs. Doxorubicin loaded zein nanoparticles inhibited tumor growth and enhanced survival rate in mouse bearing JC tumors. A novel amphiphilic polymer, PEG-Zein was synthesized using zein and polyethylene glycol. The amphiphilic PEG-Zein self-assembled into nanoscopic micelles and the size of micelles ranged from 50 to 165 nm. Zein which is water insoluble protein formed the hydrophobic core, while PEG, a water soluble polymer formed the hydrophilic shell. The PEG-Zein micelle was stable in physiological pH (7.4 ), with a CMC of 0.025±0.0095 g/L. In immunogenicity studies of PEG-Zein micelles, no antibodies were produced. A model hydrophobic compound curcumin was encapsulated in PEG-Zein micelles. Curcumin loaded PEG-Zein micelles had a mean diameter, polydispersity index, zeta potential, and were 124±4 run, 0.25±0.03, and -6.5±5 respectively. The encapsulation efficiency of curcumin in PEG-Zein micelles was 95±4%. Dialysis method produced smaller sized curcumin micelles with high encapsulation efficiency in comparison to film method. Further the aqueous solubility of curcumin was increased by 2000 fold when encapsulated in PEG-Zein micelles. Release of curcumin was sustained for 24 hrs in citrate buffer pH 7.4.Doxorubicin was loaded using film and dialysis methods into PEG- Zein micelles. The mean diameter and polydispersity index of doxorubicin loaded PEG-Zein micelles were 153±3 run, and 0.18±0.06 respectively. The encapsulation efficiency of doxorubicin was 92±6 %. The doxorubicin solubility in PEG-Zein micelles was enhanced by 1000 fold in phosphate buffer pH 7.4. In vitro release of doxorubicin from PEG-Zein micelles was sustained for 24 hrs. There was 2 to 4 fold reduction in IC 5o values of doxorubicin from PEG-Zein micelles in Pgp overexpressing cancer cell lines. There was enhanced cell uptake and cell retention of doxorubicin from PEG-Zein micelles. Inhibition of calcein AM efflux in Pgp overexpressing resistant cell lines demonstrated that PEG-Zein has P-gp inhibitory activity. Further PEG-Zein micelles resulted in enhanced biodistribution of doxorubicin to tumors and significantly lower amount in other organs. Doxorubicin loaded PEG-Zein micelles inhibited tumor growth and enhanced survival rate in mouse bearing allograft JC tumors compared to free doxorubicin. Overall the results from this study demonstrate the potential of zein based nanocarriers for drug delivery application

Abstract 5709: Formulation development and evaluation of nanostructured lipid carriers for topical delivery of honokiol

Abstract Purpose: Honokiol, a plant lignan isolated and purified from Magonila Officinalis has been shown to have chemopreventive effects in chemically induced and UV-B induced skin tumor development in SKH-1 mice. However poor solubility of honokiol greatly limits its clinical application. The key objectives of the present investigation are to improve the solubility and stability of honokiol by encapsulating in nanostructured lipid nanoparticles. Methods: Honokiol was incorporated into nanostructured lipid nanoparticles and cream using hot homogenization method. Compritol 888 ATO, Precirol ATO 5, and miglyol 812 as lipid matrices and poloxamer 188 as emulsifier. Lipid nanoparticles were characterized for size, zeta potential, shape and morphology using photon correlation spectroscopy (PCS) and atomic force microscopy (AFM). Honokiol was quantified by isocratic HPLC method using mobile phase of 60:40 (%v/v, acetonitrile:water) at a flow rate of 1 ml/min at a wavelength of 254 nm. Permeation studies were carried out using porcine dermatome skin in Franz diffusion cell. Honokiol concentration corresponding to 0.5 mg/ml in sesame oil, cream and lipid nanoparticles was dispersed in PBS pH 7.4. Permeation studies were performed till 24hrs; the samples (200 µl) were withdrawn at regular time intervals and replaced with fresh medium. Amount of honokiol permeated across porcine skin was quantified using HPLC method. Stability of cream and lipid nanoparticle formulations was studied at 30° C and 65 % relative humidity in a stability chamber for one month. Results: The size of the optimized honokiol loaded lipid nanoparticles was 145±7 nm with a polydispersity index of 0.24±0.02. The zeta potential and encapsulation efficiency was 0.34±0.02 mV and 97±2 %. The particle size of lipid nanoparticles measured in AFM was very close to size measured by PCS. In case of precirol cream, particle size was more than 20 µm with drug content of 89±5 %. Honokiol loaded nanostructured lipid nanoparticles, and cream were stable for about 30 days without any significant changes in particle size, and drug content at 30° C and 65 % relative humidity. Skin treated with honokiol entrapped in nanoparticles and cream has shown more retention of honokiol in the skin compared to plain honokiol in sesame oil. Proposed lipid cream and nanoparticles formulation has shown effective skin penetration of honkiol. From the data it is evident that nanoparticles has shown higher amount of honokiol in skin compared to cream formulation. Conclusions: Preliminary results of this study have shown the potential of lipid based formulations for topical delivery of honokiol. Chemopreventive potential and efficacy of honokiol encapsulated in lipid based formulations will be evaluated in UV-B induced skin cancer model. Acknowledgements: Supported by Translational Cancer Research Center funded by the South Dakota Governor's Office of Economic Development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5709. doi:1538-7445.AM2012-5709</jats:p

Synthesis of Novel Biodegradable Methoxy Poly(ethylene glycol)–Zein Micelles for Effective Delivery of Curcumin

Novel biodegradable micelles were synthesized by conjugating methoxy poly­(ethylene glycol) (mPEG) to zein, a biodegradable hydrophobic plant protein. The mPEG–zein micelles were in the size range of 95–125 nm with a low CMC (5.5 × 10–2 g/L). The micelles were nonimmunogenic and were stable upon dilution with buffer as well as 10% serum. Curcumin, an anticancer agent with multiple delivery challenges, was encapsulated in mPEG–zein micelles. The micelles significantly enhanced the aqueous solubility (by 1000–2000-fold) and stability (by 6-fold) of curcumin. PEG–zein micelles sustained the release of curcumin up to 24 h in vitro. Curcumin-loaded mPEG–zein micelles showed significantly higher cell uptake than free curcumin in drug-resistant NCI/ADR-RES cancer cells in vitro. Micellar curcumin formulation was more potent than free curcumin in NCI/ADR-RES cancer cells, as evidenced from the 3-fold reduction in IC50 value of curcumin. Overall, this study for the first time reports a natural protein core based polymeric micelle and demonstrates its application for the delivery of hydrophobic anticancer drugs such as curcumin