Nanotechnology in oncology: Characterization and in vitro release kinetics of cisplatin-loaded albumin nanoparticles: Implications in anticancer drug delivery.

CONTEXT: Nanotechnology is an empowering technology that holds promise in cancer therapeutics by increasing the ratio of tumor control probability to normal tissue complication probability. It can increase the bioavailability of the drug at the target site, reduce the frequency of administration and reach otherwise lesser-accessible sites. The present study shows the feasibility of the cisplatin-loaded albumin nanoparticle as a sustained delivery system. AIMS: Cisplatin is one of the most widely used chemotherapeutic agents for the treatment of malignant disorders. Conventional cisplatin formulation given as intravenous infusion has low bioavailability to the target organ in addition to significant side-effects, like ototoxicity and nephrotoxicity. The aim of this study was to develop a protein-based nanoparticulate system for sustained release of cisplatin.
MATERIALS AND METHODS: Nanoparticles were prepared by the coacervaton method of microcapsulation and chemical cross-linking with glutaraldehyde. Particle size was characterized by dynamic light scattering and transmission electron microscopy. RESULTS AND
CONCLUSIONS: Using the coacervation method, nanoparticles of less than 70 nm diameter were produced. Drug encapsulation measured by ultraviolet spectroscopy varied from 30% to 80% for different ratios of cisplatin and protein. In vitro release kinetics shows that the nanoparticle-based formulation has biphasic release kinetics and is capable of sustained release compared with the free drug (80% release in 45 h). The study proves the feasibility of the albumin-based cisplatin nanoparticle formulation as a sustained release vehicle of cisplatin.

Introduction

Cis-diamminedichloro platinum (CDDP, henceforth cisplatin) is one of the common chemotherapeutic agents used in the treatment of cancer.[1] It is widely used for the treatment of head and neck cancer,[2] cervix cancer,[3] upper gastrointestinal malignancies (esophagus and stomach),[4] lung cancer[5] and bone sarcomas.[6] Clinically, cisplatin is used as part of concurrent chemoradiotherapy (in radisentizing dose for head and neck and cervical cancer) or as a part of combination chemotherapy in lung cancer and gastric cancer. However, at therapeutic concentrations, the drug is characterized by significant side-effects like nephrotoxicity, ototoxicity, mucositis and myelosupression, often leading to life-threatening septicemia.[7] In addition, a significant percentage of the drug is excreted without reaching the target organ. The effect of the drug in conventional formulations is short lasting. Therefore, to maintain the concentration of the drug in the therapeutic window, frequent doses of administration are required. This explains why cisplatin is administered weekly at a dose of 40 mg/m2 during radiotherapy.[8] This also increases the chance of cumulative toxicity.

A clinically viable formulation that is capable of maintaining the concentration of the drug in the plasma within the therapeutic window for prolonged duration will be highly useful as it will avoid frequent doses of administration, will reduce the side-effects and, therefore, will increase the patient compliance. This concept is pictorially depicted in Figure 1, which shows the benefit of the nanoparticle-based drug delivery system to maintain the concentration of the drug in the therapeutic window for a longer duration.

Figure 1

Comparison of the pharmacokinetic profile of conventional drug formulation (continuous line) and nanoparticles (dotted line). Nanoparticles are postulated to maintain the concentration in the therapeutic window for a longer duration

Nanotechnology is an empowering technology with promising applications in the drug delivery system. In view of a higher surface to volume ratio compared with the same material in a bulk form, nanoparticles can entrap a larger amount of drug. Particles of less than 100 nm can escape phagocytosis and reach otherwise lesser-accessible sites.[9] The drug release from polymeric nanoparticles is related to the degradation of the matrix; therefore, it can achieve sustained drug release and, because of their small size, they can achieve higher concentrations at relatively lesser-accessible sites. It has also been shown in the literature that malignant cells actively take up nanoparticles compared with normal cells.[10] Many polymeric nanoparticles have been evaluated in the literature.[11-15] Recently, albumin nanoparticles have gained a wider acceptance clinically as they are biocompatible and biodegradable. Albumin is the main component of plasma protein, and most of the drugs bind to albumin.[16] Various drugs have been evaluated with albumin as nanocarriers, and one of them (paclitaxal) is already in clinical use.[17]

The aim of the present research was to develop an albumin nanoparticle-based sustained release system for cisplatin. The paper describes the preparation, characterization and in vitro release kinetics of the drug from albumin-bound cisplatin nanoparticles and their prospect of application as a sustained release delivery system of cisplatin for anticancer therapy and various other applications.

Materials and Methods

Materials

The following commercially available chemicals and accessories were used as received: bovine serum albumin (BSA; Spectrochem, Mumbai, India), 25% glutaraldehyde (Spectrochem, Mumbai, India), cisplatin (Pfizer, Mumbai, India), ethanol (Merck, Mumbai, India), mannitol (Ranbaxy, Mumbai, India), phosphate buffer (prepared from NaH2PO4 and Na2HPO4 obtained from Qualigen Fine Chemicals, Mumbai, India) and cellulose ester membrane (Spectrum Laboratories, Chennai, India).

Preparation of Drug-Loaded Nanoparticles

BSA nanoparticles were prepared by the coacervation method following two different processes, differing in the step where the drug and the chemical cross-linking agent are added.[1314]

In model A, 16 ml of ethanol was added drop wise to 8 ml of the aqueous solution of BSA (2% w/v) under continuous stirring until the solution became turbid. Coacervate thus obtained was hardened for 2 h with glutaraldehyde (1.56 μg/mg protein). This was later purified by centrifugation (Backman J-22) at -4°C. Three batches of nanoparticles were prepared using speeds of centrifugation corresponding to 45000g, 30000g and 20000g forces. The supernatants were removed and the pellets were suspended in phosphate buffer (PBS; pH 7.4) containing cisplatin and incubated for 4 h. The drug protein ratios used were 1:8 and 1:16. Then, cisplatin-loaded nanoparticles were separated from the free drug by centrifugation (10000 rcf/45 min) at 4°C and the samples were lyophilized with mannitol (2% w/v) at -48°C and 28 × 10-3 M Bar pressure for 24 h [Figure 2a].

Figure 2

(a) Schematic diagram of the synthesis of nanoparticles (model A). (b) Schematic diagram of the synthesis of nanoparticles (model B)

In model B, cisplatin (drug protein ratios 1:8 and 1:16) was firstly incubated with 10 ml of an aqueous solution of BSA (2% w/v) for 4 h. The pH of this solution was adjusted with 1M HCl to the isoelectric pH of the protein (pH 5.5). Aqueous phase was dissolved with ethanol (ratio, water to ethanol, 1:2, v/v). The coacervate so formed was hardened for 2 h with glutaraldehyde (1.56 μg/mg protein), and the resulting nanoparticles were purified by centrifugation at -4°C. Three batches of nanoparticles were prepared using three different speeds corresponding to 20000g, 30000g and 45000g forces. The pellet of the nanoparticles was suspended in phosphate buffer (pH 7.4; 0.1M), and each sample was lyophilized with mannitol (2%, w/v) at -48°C and 28 × 10-3 M Bar pressure for 24 h [Figure 2b].

Unloaded albumin nanoparticle controls were also prepared by both the procedures under identical conditions.

Determination of Particle Size

Lyophilized nanoparticles were suspended in PBS to make a 1% solution. Then, the particle size was measured by dynamic light scattering (DLS) protein solution dynopro 99.

Transmission Electron Microscopy

Lyophilized nanoparticles were suspended in 0.1 M sodium cacodylate buffer (pH 7.4) and a drop of aqueous solution of lyophilized powder (5 mg/ml) was placed on the grid surface, followed by staining with 1% uranyl acetate. After 5 min, excess fluid was removed and the grid surface was air dried at room temperature before loading in the microscope.

Determination of Drug Entrapment

For determination of drug entrapment, the amount of drug present in the clear supernatant after centrifugation (w) was determine by UV-spectrophotometry. A standard calibration curve of concentration versus absorbance was plotted for this purpose. The amount of drug in the supernatant was then subtracted from the total amount of drug added during the coacervation process (W). In effect, (W-w) will give the amount of drug entrapped in the pellet. Then, percentage entrapment is given by

Study of Release Kinetics

in vitro release kinetics was studied in a specialized diffusion chamber with two compartments, each compartment of volume 20 cc. The two compartments communicate through an opening of 2 cm diameter. The opening was covered with a semipermeable membrane (cut-off 3500 Da). This prevented albumin (molecular weight 66432 Da) from passing across the membrane. Each experiment was performed under constant slow magnetic stirring. Twenty milligram of drug-loaded nanoparticles, suspended in 20 ml phosphate buffer (pH 7.4), was placed in the donor compartment and the other compartment was filled with phosphate buffer of equal volume. To determine the concentration of cisplatin in the receiving compartment, samples (1 ml) were withdrawn at pre-fixed time intervals and the absorbance was measured by UV spectrophotometry at a wavelength of 300 nm. After each measurement, the same amount of phosphate buffer was reintroduced into the receiver chamber (concentration change was accounted for). Finally, the concentration corresponding to the absorbance was determined from the concentration vs. absorbance calibration curve.

Results

Physical Properties and Entrapment Efficiency

Table 1 shows the particle diameter and entrapment efficiency of the nanoparticles prepared using various centrifugation speeds and drug protein ratios. From the table, it is evident that the particle diameter ranged from 30.69 to 66.96 nm in model B and from 45.2 nm to 68.9 nm in model A. Entrapment efficiency in model A was 43–99% and in model B, this was 34–94%. Stable nanoparticles of less than 70 nm were obtained by both the methods.

Table 1

Characteristic of nanoparticles

Microscopically, monomorphic discrete particles were found in sheets and clusters of diameter 70 nm [Figures 3 and 4].

Figure 3

Transmission electron micrograph of cisplatin-loaded nanoparticles (model B) of size 55 nm and magnification 30X

Figure 4

Transmission electron micrograph of cisplatin-loaded nanoparticles (model A) of size 48.5 nm and magnification 70X

Analysis of variance (ANOVA) reveals centrifugation force to be an important determinant of particle size in model A and entrapment efficiency in model B. No such significant result was obtained by AVOVA test taking drug–albumin ratio as the independent variable (P-values not significant). It appears from the result that in model A, centrifugation force significantly affects the particle diameter, and the lowest diameter is obtained at 20000g. Therefore, we selected the model A sample prepared with 20000g and 30000g, with drug protein ratio 1:8 for this release kinetics study. The batch also had a high entrapment in addition to particle diameter of less than 70 nm.

Study of In Vitro Release Kinetics

Total cumulative release at pre-fixed time intervals in terms of percentage entrapment was calculated and plotted against time. Unlike the free drug, which peaks at 7 h and saturates fast, nanoparticles are capable of releasing the drug for more than 50 h. Therefore, this system overcomes the burst effect of the free drug. It is also found from the plot that the release kinetics is biphasic. The early phase corresponds to the release of physically bound drug to the surface of the protein, and the delayed phase is because of release of covalently bound drug from the disintegration of nanoparticles [Figure 5].

Figure 5

Release kinetics of the nanoparticle formulation compared with the free drug (formulation used model A – 30,000g and 20,000g; ratio 1:08

Discussion

Albumin is widely used for preparing nanoparticles. Both BSA[14] and human serum albumin have been used. As a major plasma protein, albumin has a distinct edge over other materials used for nanoparticle preparation. Pharmacokinetic properties of various drugs with albumin are well characterized. Being a plasma protein, it is both bioacceptable and biodegradable. Moreover, it is cheap and easily available. The process of preparing nanoparticles from albumin is not complicated, and particle size can be controlled by various factors during preparation. Drugs entrapped in albumin nanoparticles can be digested by proteases and drug loading can be quantified. Although various polymers have been used to prepare cisplatin nanoparticles,[18-25] protein-based nanoparticle preparation of cisplatin is completely novel and is a viable option for sustained drug delivery. We prepared albumin-loaded cisplatin nanoparticles of less than 70 nm diameter with high entrapment efficiency using the coacervation technique. Using centrifugation speeds corresponding to 20000g force and 30000g force, less than 60 nm particles with high entrapment efficiency of more than 90% were obtained. This system shows a sustained drug delivery 50% release at 18 h compared with the free drug, which shows an 80% release at 5 h. Albumin, an integral component of plasma protein, is biocompatible and biodegradable. Such protein-based nanoparticles are attractive vehicles for anticancer drug delivery. In view of the ultra small size, these particles would be capable of reaching otherwise lesser-accessible sites; considering the nanoparticles are taken up more avidly by malignant cells, the bioavailability of the drug is likely to be higher in tumor cells. Because of sustained release and higher surface to volume ratio, this would translate into less-frequent doses of administration and reduced toxicity. Therefore, cisplatin-loaded albumin nanoparticles are promising agents for a sustained drug delivery system in the tumor site, which would increase bioavailability. Clinically, this translates to a less-frequent dosage of administration and reduced dose. This may reduce the adverse side-effects of cisplatin, like ototoxicity and nephrotoxicity, which can be investigated in future research. In view of the particles being very small (<50 nm) and having sustained release kinetics and biocompatible albumin base, they may also be investigated as candidates for transarterial chemo embolisation for hepatocellular carcinoma and liver metastasis.