Colloids Surf B Biointerfaces. 2017 Apr 1;152:393-405. doi: 10.1016/j.colsurfb.2017.01.044.

Redox responsive albumin autogenic nanoparticles for the delivery of cancer drugs 
 

Thirupathi Kumara Raja, S., Prakash, T. and A. Gnanamani*

Biological Material Laboratory, Microbiology Division, CSIR-CLRI, Adyar, Chennai 20, Tamil Nadu, India

 

*-Author for correspondence

Dr. A. Gnanamani

Biological Material Laboratory

CSIR-CLRI, Adyar,

Chennai 600 020,

Tamil Nadu, India

Email: gnanamani3@gmail.com

 

Abstract

The present study explores preparation and characterization of redox sensitive albumin autogenic nanoparticles (ANPs) for drug delivery applications. Human serum albumin nanoparticles are prepared by desolvation method. The particles are stabilized through self-crosslinking and no external stabilizers are involved in the preparation. ANPs are then subjected to Camptothecin (CPT) drug loading. Experiments on in vitro and in vivo release profile, cytotoxic and cytocompatability, hemocompatability, blood clearance, tracking and bio imaging are studied in detail. The redox sensitive and drug release properties of ANPs studied in the presence of glutathione. Results on the physical, chemical and instrumental characterization warrant the property of the nanoparticles. ANPs obtained in the present study is biocompatible, biodegradable, effectively entangle the chosen drug, release the drug in the controlled manner, sensitive to reducing environment, nil toxicity and appreciable uptake by cells. In the current scenario on the requirement of a drug carrier with redox sensitive property to encounter cancer cells, the results of the present study on albumin nanoparticles with redox sensitivity is smart and pave the way in the cancer therapeutics.

Keywords: autogenic material, Serum albumin, oxidative refolding, nanoparticles, drug delivery

 

Supplement

In our earlier work, we have witnessed the transformation of reduced serum albumin solution to a hydrogel upon aerial oxidation. This paves way for the present study by implementing the same principle through exploring the potential use of redox chemistry in the preparation of serum albumin nanoparticles for therapeutic delivery application.

In the past few years, clinicians have observed that notable applications on therapeutic delivery and diagnostics have been done through different types of nanoparticles. Various materials have been employed by different researchers using natural and synthetic monomers and polymers to design nanoparticles, of which proteins plays an attractive role. Synthesis of these biomolecules does not necessitate harsh chemical reactions and their inherent biocompatibility and biodegradability are their major advantage. Further, protein posses various functional groups which can be utilized for surface modification and targeting of ligands. However, the major disadvantage of using protein based nanoparticles is the use of crosslinking agents for stabilization. Glutaradehyde is widely used as a crosslinking agent to stabilize the particle structure and to hold the drug. However, the use of glutaraldehyde has been restricted by FDA (US) because of its potential toxicity during degradation.

To address the said problem, the present study explores an alternative solution by exploiting the inherent functional groups of the protein and involved in the crosslinking. Despite of primary amines, hydroxyl, carboxyl and aromatic functionality of proteins, thiols (-SH) receives wide attention because of its disulphide forming capability through mild oxidation and further it does not involve any additional agents to initiate this. Though proteins like collagen, fibrin or elastin has been widely explored for its potential biomedical application and these proteins are not facilitated with the disulphide functionality. Further, primary source for these proteins purely depends on the non human species, which creates biocompatibility and immunocompatibility issues. Harvesting protein from a live human tissue is not possible unless if it is from blood, since blood has several fast rejuvenating proteins like serum albumin. Harvesting serum albumin from human blood is highly possible and it is autogenic too.

In general, when a disulphide bridge is reduced the thiol (SH) functional moiety is exposed which upon oxidation forms a disulphide bridge. However in our earlier work, we observed the non native disulphide bridge formation during protein refolding, results in a crosslinked material. This methodology has been used to develop albumin nanoparticles using traditional desolvation technique.

In brief, serum albumin is a polypeptide made of 568 amino acids in a single chain with 17 disulphide bridges and one free thiol group. In the presence of reduced glutathione, the protein undergoes thiol disulphide interchange reaction. During reduction, the reaction condition was maintained above the physiological pH, where, thiols in the reduced glutathione form a thiolate anion and act as a strong nucleophile, which readily undergoes nucleophilic attack along the disulphide bridges of the protein. The cleaved disulphide bridges of the serum albumin readily undergo oxidative refolding, when the reduction environment is removed from the system. This was achieved through desolvation method, were the reduced proteins were slowly treated with the constant addition of ethanol under vigorous stirring. The following schematic representation infers the reduction of serum albumin, reoxidation and the formation of non-native disulphide bridges results in nanoparticles formation (named as ANP –Albumin nano particle). In general, during reoxidation of serum albumin, the reduced molecule undergoes oxidative refolding and able to retains the disulphide bridges. However, it’s not necessary to retain its original conformation. Scheme 1 exhibits, pictographical representation of the ANPs formation and stabilization.

 

 

Scheme.1  Non- native form of disulphide bridge formation in serum albumin during reduction and reoxidation process. Here ‘n’ represent the number of moles of serum albumin; ‘x’ represent the number of thiol groups, under complete reduction the value of x will be 35; ‘y’ represents the number of disulphide bridges under non-native form, the value of y may be in the range of 1 to 17.

 

Results from the study conclude that the prepared albumin nanoparticles (ANPs) have the dimension in the range of 130 to 200 nm. Fig.1 a,b&c shows the Scanning electron microscope, transmission electraon microscope and Atomic force microscopic images of ANPs respectivley. It has been observed that the surface of the ANPs is sperical and smooth with an average diameter of 120 nm in size. Similarly, TEM and AFM image projects , the ANP surface morphology with the predicted size of 70-120nm.

To make sure the cellular uptake prepared ANPs, we bioconjugated the ANPs with FITC (Fluorescein isothiocyanate) to give fluorescence property. To confirm it the prepared FITC conjugated ANPs exposed to blue light which results in the green emission (Fig. 1d), further the FITC conjugated ANPs exposed to the MG-63 cell line to study the cellular uptake. The cells were observed under fluorescence microscope with blue filter to observe the ANPs. Results (Fig. 1e) confirm the cellular uptake FITC-conjugated ANPs. From the observed results, it has been concluded that the prepared nanoparticles has the capability to enter the cancer cells, which finds potential application in carrying cancer therapeutics.

Further, we have confirmed, the delivery of cancer therapeutics using model drug CPT (Camptothecin). During the preparation of ANPs, the pre dissolved CPT in the solvent mixed and at the time of desolvation CPT has been entrapped in ANPs. Further, using MG-63 cell line, we have confirmed the potential of CPT-ANPs as an effective carrier for anti-cancer therapeutic. To further elucidate, the in vivo blood clearance of the ANPs, we have conjugated fluorescence probe to the ANPs and injected into the blood stream through the rat tail and performed in vivo animal imaging (Fig. 1f). At scheduled time interval, we have analyzed the presence of ANPs in the blood through HPLC analysis. Results revealed that blood circulation half-time (t1/2) of injected nanoparticles was 12.0 h indicating relatively average circulation and therefore suitable for targeted drug delivery applications. Scheme 2 illustrates the drug loading and the interaction of drug loaded ANPs in cancer cells.

In addition to these studies, we have performed complete blood profiling analysis to further elucidate the in vivo pharmacokinetic analysis of the CPT-ANPs. Further, we have initiated to study the real time studies on the delivery of anticancer drugs in in vivo rat cancer models.

 

Acknowledgements

The first author thanks Council of Scientific and Industrial Research, New Delhi, India, for the financial assistance in the form of Research Associateship. Authors thank TRPVB, Chennai for bioimaging facility.

 

 

Fig. 1 (a) scanning electron microscope of ANPs (b) Transmission electron microscope of the ANPs (c) Atomic force microscope of the ANPs (d) FITC conjugated albumin nanoparticles emitting green light under blue light exposure (e) MG-63 intra cellular uptake of FITC conjugated ANPs observed under fluorescence microscope with blue excitation, (f) in vivo live imaging of ANPs injected albino rats observed under different time intervals.

 

 

Scheme 2 – Schematic representation of the preparation ANPs, drug loading and targeted delivery for therapeutic application for cancer.

 

Contributors

Dr. A. Gnanamani, Principal Scientist, Microbiology Division, Biological Materials Laboratory, CSIR-CLRI, Adyar, Chennai. The major research area is biomaterial development for human health care.
Dr. S. Thirupathi Kumara Raja, Research Associate, Microbiology Division, Biological Materials Laboratory, CSIR-CLRI, Adyar, Chennai. The major research area is biomaterial development for human health care.
Mr. T. Prakash, M.Pharm, Pharmacist , Microbiology Division, Biological Materials Laboratory, CSIR-CLRI, Adyar, Chennai. The major research area is Pharmaceutical product development care.