Development, characterization, and toxicity evaluation of amphotericin B–loaded gelatin nanoparticles

Available online at www.sciencedirect.com

Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 252 – 261 www.nanomedjournal.com

Original Article: Toxicology

Development, characterization, and toxicity evaluation of amphotericin B–loaded gelatin nanoparticles Manoj Nahar, MPharm, Dinesh Mishra, MPharm, Vaibhav Dubey, MPharm, Narendra Kumar Jain, PhD⁎ Pharmaceutics Research Laboratory, Department of Pharmaceutical Science, Dr. H.S. Gour University, Sagar, India

Abstract

Our aim in the present investigation was to develop a nanoparticulate carrier of amphotericin B (AmB) for controlled delivery as well as reduced toxicity. Nanoparticles of different gelatins (GNPs) (type A or B) were prepared by two-step desolvation method and optimized for temperature, pH, amount of cross-linker, and theoretical drug loading. AmB-loaded GNPs were characterized for size, polydispersity index (PI), shape, morphology, surface charge, drug release, and hemolysis. The developed GNPs (GNPA300) were found to be of nanometric size (213 ± 10 nm), having low PI (0.092 ± 0.015) and good entrapment efficiency (49.0 ± 2.9%). All GNPs showed biphasic release characterized by an initial burst followed by controlled release. The in vivo hematological toxicity results suggest nonsignificant reduction (P N .05) in hemoglobin concentration and hematocrit. Nephrotoxicity results showed that there was a nonsignificant (P N .05) increase in blood urea nitrogen and serum creatinine levels. The results confirm that developed GNPs could optimize AmB delivery in terms of cost and safety, and type A gelatin with bloom number 300 was found suitable for such preparation. © 2008 Elsevier Inc. All rights reserved.

Key words:

Nanoparticles; Gelatin; Amphotericin B; Desolvation; Nephrotoxicity

Administration of amphotericin B (AmB), a potent fungal agent, is limited by its pronounced side effects (e.g., chills, fever, nausea, hemolytic toxicity, and nephrotoxicity). 1 Conventional micellar solutions of AmB with deoxycholate (Fungizone) has been reported to cause serious nephrotoxicity. 2 The alternate novel lipid-based nanoparticulate formulations, including liposomes (AmBisome), AmB-lipid complex (Abelcet), and AmB colloidal dispersion (Amphocil), have been developed to enhance the therapeutic index and to reduce the hemolytic and nephrotoxic side effects, but are quite expensive. 3 Recently, low-priced AmB disc

Received 11 December 2007; accepted 24 March 2008. The authors are thankful to the Indian Council of Medical Research, New Delhi for financial support. ⁎Corresponding author. Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar 470003, India. E-mail address: [email protected] (N.K. Jain).

formulations containing cationic lipid dioctadecyldimethyl have been developed, but such formulations have been reported to possess low drug-loading capacity. 4,5 Moreover, AmB is the drug of choice for diseases such as visceral leishmaniasis wherein long-term infusion is needed and that require hospitalization, increased cost, and poor patient compliance. All these factors warrant the need of developing an alternative drug carrier, which may provide safe, effective, and controlled delivery of AmB. Nanoparticulate carriers have always been attractive on account of their size and capacity for spatial and temporal controlled delivery of bioactives. 6 Nanoparticles of various polymers like poly(lactide-coglycolide), poly(ɛ-caprolactone), and more recently chitosan-dextran with AmB have been investigated. 7-10 Gelatin nanoparticles (GNPs) have recently been developed to offer one good viable option because of their low cost, biocompatibility, biodegradability, low antigenicity, and applications in several parenteral formulations. 11 GNPs

1549-9634/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2008.03.007 Please cite this article as: M. Nahar, D. Mishra, V. Dubey, N.K. Jain, Development, characterization, and toxicity evaluation of amphotericin B–loaded gelatin nanoparticles. Nanomedicine: NBM 2008;4:252-261, doi:10.1016/j.nano.2008.03.007.

M. Nahar et al / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 252–261

have been previously reported for delivery of different drugs like methotrexate, 12,13 doxorubicin, 14 cycloheximide, 15 paclitaxel, 16 and chloroquine phosphate 17as well as for gene delivery. 18-21 Furthermore, antibody-modified GNPs were used to target leukemic cells and primary T lymphocytes. 22,23 Various methods, including nanoencapsulation, 24 emulsification-solvent evaporation, 13 desolvation, 25 and coacervation-phase separation, 16 have been used to prepare GNPs. GNPs prepared by many of these methods were found to be large in size and have a high PI due to heterogeneity in molecular weight of the gelatin polymer. Variation in size and PI of novel nanoparticulate carrier systems may affect drug release, drug loading, and alternation in in vivo parameters like uptake by macrophages. Moreover, poor aqueous solubility and self-aggregation of AmB present a great challenge to the development of an effective and safe carrier system. These challenges prompted us to design a nanoparticulate delivery system using a low-cost polymer like gelatin, with proper control of the process parameters that may develop a carrier with small size, low PI, and maximum entrapment efficiency (EE), which can effectively deliver AmB with fewer or no side effects. Therefore, in the present investigation we attempted to develop an alternative delivery system that may overcome the severe side effects of existing AmB formulations by reducing self-aggregation, improving patient compliance by controlled delivery, and providing safe and effective delivery at low cost as an alternative to lipid-based formulations.

Methods Materials Gelatin type A from porcine skin (300, 175 bloom), gelatin type B from bovine skin (75 bloom), glutaraldehyde grade I (25% v/v aqueous solution), trypsin from bovine pancreas, HEPES buffer, and amberlite XAD 16 resin were purchased from Sigma Chemical Co. (St Louis, MO). Acetone, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were purchased from Central Drug House (New Delhi, India). AmB was a gift from Dabur India Ltd. (Ghaziabad, UP, India). All other chemicals were of analytical grade and used as received. Triple-distilled deionized water was used for all experiments. Preparation of GNPs GNPs were prepared by a two-step desolvation method reported by Coester et al 25 with slight modifications. Briefly, 200 mg gelatin (A or B) was dissolved in distilled water (10 mL) under constant heating at 40° ± 1°C. Acetone (10 mL) was added to the gelatin solution as a desolvating agent to precipitate the high-molecular-weight (HMW) gelatin. The supernatant was discarded, and the HMW gelatin was redissolved by adding distilled water (10 mL) with stirring at 600 rpm (Remi, Mumbai, India) under

253

constant heating. The pH of the gelatin solution at the second desolvation step was adjusted (between 2 and 12). AmB (dissolved in 500 μL of DMSO) was added in aqueous polymer phase, followed by dropwise addition of acetone (30 mL) to form GNPs. At the end of the process, glutaraldehyde solution (25% v/v aqueous solution) was added as a cross-linking agent, and the solution was stirred for 12 hours at 600 rpm. The unentrapped AmB was removed by adding 10 mL of adsorbent polymer beads (2% w/v), amberlite XAD 16 followed by filtration 7 (1-μm filter; Whatman Japan KK, Tokyo, Japan). DMSO was removed with repeated mild washing with distilled water. The GNPs obtained were lyophilized with trehalose (5% w/v solution) for further investigations. Effect of parameters like pH, temperature, amount of glutaraldehyde, and theoretical drug loading on the size, PI, and EE of the GNPs was studied. Determination of EE, yield, and actual drug loading Entrapment efficiencies of GNPs were determined by the method reported by Leo et al 26 Briefly, AmB-loaded nanoparticles (10 mg) were dispersed in an aqueous solution (10 mL; NaCl, 0.9%, w/v and 5% v/v DMSO) containing trypsin (200 μg/mL) at a trypsin-to-nanoparticles ratio of 1:5 (w/w). The dispersion was kept for 5 hours at 37° ± 1°C in the dark under magnetic agitation; a clear solution was obtained, and free AmB was quantified using highperformance liquid chromatography by the method described by Echevarrya et al, 27 with minor modifications. The estimation was carried out using a C18 column (250 × 34.6 mm, 5 mm particle size) (Shimadzu, Kyoto, Japan), preceded by a guard column (45 × 34.6 mm). The mobile phase was composed of acetonitrile–acetic acid (1%)–water (41:43:16) at a flow rate of 1.5 mL/min. The chromatography was carried out at room temperature (25° ± 1°C). The ultraviolet (UV) detection was performed at 405 nm using an SPD-M10Avp diode array UV detector (Shimadzu, Kyoto, Japan). The retention time was found to be at 4.3 minutes. The EE was calculated as follows: Drug entrapment efficiencyðEEÞðkÞ ¼ ðmass of drug in GNPs=mass of drug used in formulationÞ
100 ð1Þ

The purified nanosuspension was ultracentrifuged (L-8 60M; Beckman, Buckinghamshire, UK) at 31,000 g for 1 hour at 4° ± 1°C. The supernatant was discarded and the pellet was freeze-dried. The yields of various GNPs were calculated using Eq. (2), whereas the actual drug contents of GNPs were calculated using Eq. (3). Nanoparticles yieldðkw=wÞ ¼ ðmass of recovered GNPs=total mass of polymer and drug addedÞ
100

Actual drug loadingðkw=wÞ ¼ ðmass of drug in GNPs=mass of GNPs recoveredÞ
100

ð2Þ

ð3Þ

254

M. Nahar et al / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 252–261

Particle size, shape, surface charge, and PI The particle size, surface charge, and PI were determined by using a Malvern Zetasizer (NanoZS; Malvern Instruments Inc., Worcestershire, UK). Particle size and PI of GNPs were determined by light-scattering method based on laser diffraction at an angle of 135 degrees. Typically, GNPs were suspended in aqueous medium (1.5 mL) and placed in a cuvette at a concentration of 0.3 mg/mL at 25° ± 1°C. The viscosity and refractive index of the continuous phase were set to those specific to water. GNPs surface charge was determined by laser doppler anemometry. Briefly, GNPs were suspended in 1 mM HEPES buffer and adjusted to pH 7.4 by 0.1 M HCl so as to maintain a constant ionic strength. Transmission electron microscopy (TEM) (H7500; Hitachi Ltd., Tokyo, Japan) was used for determination of shape and size of GNPs. The aqueous dispersion (one drop) was placed over a 400-mesh carbon-coated copper grid followed by negative staining with phosphotungstic acid solution (3% w/v, adjusted to pH 4.7 with KOH) and placed at the accelerating voltage of 95 kV. UV-visible spectral study UV-visible (UV-vis) spectra of pure AmB as well as lyophilized AmB-loaded GNPs were obtained using UV-vis spectrophotometer (Shimadzu1601; Kyoto, Japan). Briefly, AmB-loaded lyophilized GNPs were dispersed in 5.0 mL of deionized water (containing 5% v/v DMSO) to give a final AmB concentration of 10 μg/mL. The suspension is then filtered with a 0.22-μm membrane filter and scanned in the range 200-550 against plain nanoparticles as blank. In vitro release kinetics In vitro release kinetics was evaluated by equilibrium dialysis membrane, and quantification was carried out by high-performance liquid chromatography. Briefly, 10 mg AmB equivalent of various formulations of GNPs were suspended in 2 mL of phosphate-buffered saline (PBS, pH 7.4) containing 5% v/v of DMSO in a dialysis bag (molecular weight cutoff 5 kD; AnaSpec, Inc., San Jose, CA) and dialyzed against 50 mL of PBS/DMSO (95%:5% v/v) at a speed of 50 rpm. Samples (500 μL) were collected at known intervals and volumes and were replenished by PBS/DMSO (95%:5% v/v) while maintaining strict sink conditions throughout the experiment. In vitro hemolytic study Whole human blood was collected in HiAnticlot bloodcollecting vials (Himedia, Mumbai, India). The red blood cells (RBCs) were separated by centrifugation and resuspended in normal saline solution to a 10% hematocrit value. 28 After the centrifugation (1500 g, 5 minutes at 4° ± 1°C) the supernatant was removed and the RBCs pellet was lysed with sterile water (considered as 100% hemolysis) and normal saline (taken as blank for spectrophotometric

estimation at 540 nm). Suitably diluted AmB-loaded GNPs (0.5 mL; GNPA300, GNPA175, and GNPB75) were added separately to 4.5 mL of normal saline and allowed to interact with the RBCs suspension. Similarly, 0.5 mL of drug solution and 0.5 mL of blank gelatin GNPs were mixed with 4.5 mL of normal saline and allowed to interact with the RBCs suspension. The AmB and GNPs were taken in separate tubes at equivalent concentrations. Thus, the hemolysis data of plain drug, blank GNPs, and AmB-loaded GNPs were compared. Percentage hemolysis was determined for each sample as follows: % Hemolysis ¼ABs=AB100
100

ð4Þ

where ABs is the absorbance of the sample and AB100 is the absorbance of the control (without formulation). In vivo toxicity studies In vivo studies were conducted following the protocol by the Institutional Animals Ethical Committee of the University. Proper humane care of animals was taken during study period. Various blood biochemical parameters like hemoglobin concentration, white blood cell (WBC) count, and hemocrit percentage were determined to study hematological toxicity associated with AmB-loaded nanoparticles using adult male Swiss albino mice weighing 30 to 50 gm. RBCs, leukocytes, and platelets were counted by standard methods. 29 The nephrotoxicity associated with the newly developed system was also determined in the blood samples obtained from survivor mice at day 14 by two measures of kidney function: blood urea nitrogen (BUN) and serum creatinine. Animals were divided into five groups at six animals per group (n = 6). Treatment was commenced from day 1. All groups except control (group 1) received a daily dose of 1 mg/kg body weight of AmB up to day 7. The AmB group (group 2) received daily 1 mg/kg body weight of AmB as a solution in 2% w/v in DMSO. The blank GNPs group (group 3) received 100 μL of blank nanoparticles intravenously. The drug GNPs group (group 4) received AmBloaded GNPs, whereas the AmBisome group (group 5) received liposome market preparation of AmB. Statistical analysis Statistical analysis was performed with Graph Pad Instat Software (Version 3.00; Graph Pad Software, San Diego, CA) by one-way analysis of variance followed by TukeyKramer test for multiple comparison. Difference with P b .05 was considered statistically significant.

Results GNPs were prepared by a two-step desolvation method as described before. The effects of various process variables on formulation parameters, including amount of glutaraldehyde, temperature, pH, and theoretical drug loading, were optimized. Glutaraldehyde solution (25% v/v in water) was

M. Nahar et al / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 252–261

255

Table 1 Effect of amount of glutaraldehyde on particle size, PI, and EE (at 40°C and pH 3 and 11 for type A and type B, respectively, at 2% w/w theoretical drug loading)⁎ Amount of glutaraldehyde (μL)

Size (nm), (PI) GNPA300

GNPA175

GNPB75

GNPA300

EE (%) GNPA175

GNPB75

50

412 ± 17 (0.434 ± 0.062)

498 ± 23 (0.503 ± 0.070)

514 ± 31 (0.321 ± 0.043)

43.6 ± 2.3

41.1 ± 2.1

17.9 ± 0.8

100

303 ± 12 (0.283 ± 0.095)

374 ± 34 (0.454 ± 0.082)

413 ± 49 (0.412 ± 0.070)

44.0 ± 1.4

41.9 ± 2.3

18.8 ± 1.2

200

214 ± 12 (0.066 ± 0.010)

272 ± 23 (0.189 ± 0.013)

316 ± 26 (0.228 ± 0.019)

44.9 ± 1.7

42.1 ± 2.8

20.0 ± 1.8

300

161 ± 19 (0.095 ± 0.017)

198 ± 32 (0.199 ± 0.014)

271 ± 43 (0.274 ± 0.018)

45.0 ± 2.1

42.7 ± 2.2

21.8 ± 2.2

400

47 ± 9 (0.271 ± 0.062)

80 ± 12 (0.302 ± 0.187)

99 ± 11 (0.327 ± 0.083)

45.7 ± 3.0

43.1 ± 2.4

23.4 ± 2.8

⁎Values represent mean ± SD (n = 3).

Table 2 Effect of temperature on particle size, PI, and EE (200 μL glutaraldehyde, pH 3 and 11 for type A and type B, respectively at 2% w/w theoretical drug loading)⁎ Temperature (°C)

Size (nm), (PI)

EE (%)

GNPA300

GNPA175

GNPB75 382 ± 27 (0.243 0.021)

GNPA300

GNPA175

GNPB75

40

214 ± 13 (0.064 ± 0.090)

254 ± 22 (0.132 ± 0.021)

47.8 ± 1.8

44.0 ± 2.3

22.6 ± 1.4

50

958 ± 57 (0.204 ± 0.031)

1016 ± 98 (0.323 ± .0.03)

1150 ± 110 (0.403 ± 0.024)

45.8 ± 2.2

41.3 ± 1.9

19.4 ± 1.6

60

1095 ± 30 (0.330 ± 0.045)

1160 ± 67 (0.400 ± 0.022)

1219 ± 44 (0.556 ± 0.014)

43.9 ± 2.1

40.0 ± 2.2

18.8 ± 1.4

GNPB75

⁎Values represent mean ± SD (n = 3).

Table 3 Effect of pH on particle size, PI, and EE (at 40°C and 200 μL glutaraldehyde at 2% w/w theoretical drug loading)⁎ pH

Type A

Type B

Size (nm), (PI)

EE (%)

GNPA300

GNPA175

GNPA300

GNPA175

2.0

233 ± 16 (0.140 ± 0.081)

244 ± 12 (0.232 ± 0.051)

–

45.0 ± 2.7

44.8 ± 2.4

–

3.0

213 ± 15 (0.065 ± 0.092)

230 ± 25 (0.103 ± 0.031)

–

49.8 ± 2.5

42.6 ± 2.1

–

4.0

220 ± 26 (0.110 ± 0.014)

260 ± 23 (0.11± 0.042)

–

41.6 ± 2.5

40.7 ± 1.9

–

GNPB75

10.0

–

–

400 ± 30 (0.340 ± 0.028)

–

–

20.6 ± 2.2

11.0

–

–

310 ± 23 (0.140 ± 0.021)

–

–

24.4 ± 1.9

12.0

–

–

349 ± 20 (0.235 ± 0.018)

–

–

17.6 ± 1.7

⁎Values represent mean ± SD (n = 3).

used as cross-linker for preparation of GNPs. The amount of glutaraldehyde was varied from 50 to 400 μL, while keeping other formulation variables constant (at temperature 40°C and pH 3 and 11 for type A and type B, respectively, at 2% w/w theoretical drug loading) to achieve optimized GNPs with nanometric size, low PI, and maximum EE. The results showed that reduction in particle size was directly related to the amount of glutaraldehyde used (Table 1). There was approximately five to ten times reduction in particle size, as the amount of glutaraldehyde varied from 50 to 400 μL in all GNPs (Table 1). Similarly, temperature was varied from 40° ± 1°C to 60° ± 1°C (at 200 μL glutaraldehyde, pH 3 and 11 for type A and type B, respectively, at 2% w/w theoretical drug loading). The result suggested that 40° ± 1°C was the minimum as well as the optimum temperature resulting in

GNPs with nanometric size, narrow size distribution (low PI), but no significant difference in EE (Table 2). Additionally, formulation pH at the second desolvation was also varied, and specific pH, in this case pH 3 for type A or pH 11 for type B, was found to be optimum, because at these pH values, strength of electrostatic interactions could be maximized, resulting in small particles with low PI and maximum EE while keeping other formulation variables constant (Table 3). Furthermore, theoretical drug loading was varied from to 1% to 8% w/w so as to observe the effect on size, PI, and drug EE (at a temperature of 40°C, 200 μL glutaraldehyde, and pH 3 and 11 for type A and type B, respectively). Our results revealed that 2% w/w drug loading was considered optimum, in that nanometric GNPs with maximum EE were observed and after 2% w/w there was no

256

M. Nahar et al / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 252–261

Table 4 Effect of theoretical drug loading on particle size, PI, and EE (at 40°C, 200 μL glutaraldehyde, and pH 3 and 11 for type A and type B, respectively)⁎ Theoretical drug loading (% w/w)

Size (nm), (PI)

1

201 ± 11 (0.11 ± 0.018)

244 ± 12 (0.120 ± 0.016)

309 ± 13 (0.134 ± 0.027)

2

213 ± 10 (0.092 ± 0.015)

249 ± 14 (0.149 ± 0.052)

313 ± 12 (0.240 ± 0.061)

4

219 ± 21 (0.104 ± 0.09)

266 ± 21 (0.180 ± 0.076)

322 ± 10 (0.254 ± 0.013)

8

225 ± 32 (0.114 ± 0.021)

270 ± 34 (0.149 ± 0.034)

339 ± 23 (0.249 ± 0.026)

GNPA300

EE (%) GNPA175

GNPB75

GNPA300

GNPA175

GNPB75

40.8 ± 1.8

40.5 ± 1.32

12.25 ± 2.1

49.0 ± 2.9

45.1 ± 2.6

19.6 ± 1.9

49.9 ± 3.1

45.9 ± 2.7

20.9 ± 1.8

51.0 ± 3.2

46.9 ± 3.1

21.8 ± 2.2

⁎ Values represent mean ± SD (n = 3).

Table 5 Characterization of blank and AmB-loaded (2% w/w) GNPs⁎ Formulations GNPA300 GNPA175 GNPB75

Size(nm) (PI)

Blank

204 ± 12 (0.104 ± 0.013)

AmB-loaded

213 ± 10 (0.092 ± 0.015)

Blank

234 ± 13 (0.320 ± 0.096)

AmB-loaded

249 ± 14 (0.149 ± 0.012)

Blank

304 ± 23 (0.234 ± 0.042)

AmB-loaded

313 ± 12 (0.240 ± 0.023)

EE (% w/w) – 49.0 ± 2.9 – 45.1 ± 2.6 – 19.6 ± 1.9

Actual drug loading (% w/w)

Nanoparticles yield (% w/w)

–

66.25 ± 2.42

20.6 ± 0.24

64.6 ± 1.20

21.89 ± 0.76

1.67 ± 0.32 –

Zeta potential (mV)

63.14 ± 3.26

10.32 ± 0.60

62.4 ± 2.10

11.94 ± 0.86

–

50.29 ± 3.10

–10.23 ± 1.10

0.87 ± 0.14

48.64 ± 2.60

–12.26 ± 0.98

1.40 ± 0.21

⁎ Values represent mean ± SD (n = 3).

significant (P N .05) increase in all three parameters as we increased drug loading to 8% (Table 4). The yield, practical drug loading, and zeta potential values of various blank and AmB-loaded GNPs are presented in Table 5. The TEM characterization revealed that the GNPs were spherical in shape (Figure 1). However, some variation in size distribution (PI) was observed in the TEM image, which might be attributed to an uncontrolled charge neutralization process involved between oppositely charged chains occurring during the formation of nanoparticles at specific pH, which is consistent with reports. 30,31 The UV-vis spectra of AmB-loaded GNPs and pure AmB were recorded. The AmB-loaded GNPs spectrum showed a peak with a slight shift at λmax (showed peak at 406.8, 363.2, 380 nm in methanol solution) but with decreased intensities. In vitro release of the AmB-loaded GNPA300, GNPA175, and GNPB75 for 196 hours at pH 7.4 was studied. It was observed that the drug released from the GNPA300, GNPA175, and GNPB75 were characterized by an initial burst of 39.3 ± 3.40%, 32.65 ± 1.49%, and 24.65 ± 2.56%, respectively, in the first 4 hours, followed by controlled release. The biphasic pattern of drug release observed from GNPs was similar to that reported by other research groups. 12,15 The AmB released over 196 hours from formulations GNPA300, GNPA175, and GNPB75 were 90.30% ± 1.20%, 85.24% ± 2.34%, and 74.25% ± 2.65%, respectively (Figure 2). A significantly higher (P b .05) AmB release was obtained with the GNPA300 formulation in comparison to GNPA175 and GNPB75 formulations at various time points (after 4 hours).

The in vitro hemolytic toxicity of formulations was compared with plain drug. The results depicted a 10 times reduction in hemolysis of formulations in comparison to plain AmB. The percentage hemolysis observed in various drug-loaded formulations GNPA300, GNPA175, and GNPB75 were 2.4% ± 0.6%, 3.6% ± 0.24%, and 4.89% ± 1.1%, respectively (Figure 3). Various hematological parameters like RBC counts, hemoglobin content, percentage hematocrit, and platelet count were evaluated in a mouse model at AmB equivalent to 1 mg/kg, 5 mg/kg, and 10 mg/kg for AmB solution, AmBisome, and GNPs (Table 6). The results indicate that there were significant differences (P b .05) in all blood parameters (except WBCs) for plain AmB solution (Table 6) compared with control. The AmB-loaded GNPA300 and AmBisome showed no significant decrease (P N .05) in any of these parameters except platelet count. Similarly, blank GNPs showed no significant change (P N .05) in any of these parameters except platelet count. To investigate the renal toxicity, mice were treated with AmBisome or GNPA300 (equivalent to 1 mg/kg, 5 mg/kg, and 10 mg/kg AmB). On day 8, serum from surviving mice was collected and analyzed for BUN and serum creatinine concentrations. Mice injected with all doses of plain AmB had significantly (P b .001) increased BUN and serum creatinine concentrations (Figure 4, A and B). This significant increase in these two parameters confirmed the renal toxicity caused after plain AmB administration. On the contrary, BUN and serum creatinine levels of mice receiving AmB-loaded GNPA300 or marketed formulation

M. Nahar et al / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 252–261

257

Figure 3. In vitro hemolysis of GNPs. Values represent mean ± SD (n = 3). Figure 1. TEM photomicrograph of AmB-loaded GNPA300 (110,000×).

Figure 2. Percentage cumulative release of AmB from various GNPs in PBS (pH 7.4) at 37° ± 05°C. (A), GNPA300 versus GNPA175 and GNPB75 difference is significant at all time points after 4 hours (P b .05). (B), GNPA300 versus GNPB75 difference is significant at 8, 24, and 48 hours (P b .05). Values represent mean ± SD (n = 3).

(AmBisome) showed no significant (P N .05) difference from the control group, as shown in Figure 4, A and B. Discussion Gelatin nanoparticles have the potential to be an efficient, viable, safe, and cost-effective system for administration of AmB on account of their biodegradability, biocompatibility, suitability for intravenous applications, and low immunogenicity. Different gelatins (A, B) with different bloom numbers (300, 175, and 75) were used to assess the suitable gelatin for GNPs formulations, which

would result in nanometric size, lowest PI, and maximum EE. However, heterogeneity in molecular weight of the gelatin was one major obstacle to formulating such GNPs; hence a two-step desolvation method was selected. In this method low- and variable-molecular-weight gelatin chains were discarded in the first desolvation step, and the subsequently obtained high- and uniform-molecular-weight gelatin was used for the formulation of GNPs, which ensured nanometric particles with low PI. The probable mechanism of GNPs formation by this method could be addition of a desolvating agent, leading to reduction in water available to keep the hydrated gelatin chain in solution, resulting in the agglomeration and subsequent formation of particles. 25,30 The pH of gelatin aqueous solution was adjusted before addition of AmB (in 500 μL DMSO) so as to preclude self-aggregation of AmB. This could be attributed to acquisition of specific charge by AmB at a particular pH, leading to a decrease in self-association of AmB. The possible explanation for such pH-dependent behavior is that AmB possessed two pKa values due to two ionizable groups, the amino group (pKa 5.7) and the carboxylate group(pKa 10). Any formulation effort carried out between pH 4 and 9 may lead to intermolecular electrostatic interactions between opposite-charged AmB, further leading to a high level of aggregation, as described elsewhere by several workers. 30,31 This aggregation could be a major reason for the high hemolytic and other toxicities of AmB. 32 The effect of various process variables on formulatory parameters were optimized to prepare nanometric-size GNPs with a narrow size distribution (low PI) and maximum EE. When the amount of glutaraldehyde was varied from 50 to 400 μL there was an approximately 10 times reduction in particle size. The possible explanation for particle size reduction could be attributed to crosslinking of free amine groups at the nanoparticle surface by glutaraldehyde, which caused hardening of particles

258

M. Nahar et al / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 252–261

Table 6 Hematological parameters on post-injection effects at day 8 after seven doses (1, 5, and 10 mg/kg/day of AmB) Parameters †

Doses (mg/kg/day) 6

RBCs (× 10 /μL)

Hemoglobin (g/dL)

3

WBCs (× 10 /μL)

Hematocrit (%)

3

Platelet (× 10 /μL)

Plain AmB solution

AmB-loaded GNPA300

AmBisome

Control

Blank GNPA300

1

6.03 ± 0.4⁎

7.08 ± 1.8

7.31 ± 1.3

8.57 ± 0.2

8.62 ± 1.11

5

5.12 ± 0.68⁎

6.58 ± 1.1⁎

7.06 ± 1.6

10

4.24 ± 0.45⁎

6.10 ± 0.81⁎

6.56 ± 1.0⁎

1

13.0 ± 0.8⁎

14.6 ± 2.7

16.4 ± 0.9

17.6 ± 1.3

17.5 ± 0.6

5

10.4 ± 0.3⁎

12.4 ± 1.4⁎

16.1 ± 1.4

10

09.8 ± 0.2⁎

11.0 ± 0.8⁎

15.8 ± 1.1

1

6.0 ± 0.7

6.4 ± 1.3

6.8 ± 2.7

7.1 ± 1.1

6.0 ± 1.0

5

5.8 ± 0.8

6.2 ± 0.8

6.5 ± 1.2

48.4 ± 3.4

46.7 ± 1.56

547 ± 34

129 ± 45⁎

10

5.5 ± 0.9

5.7 ± 0.4

6.2 ± 1.5

1

35. ± 3.6⁎

42.1 ± 4.6

43.9 ± 3.8

5

32.1 ± 1.3⁎

39.4 ± 3.2⁎

43.1 ± 3.2

10

30.4 ± 2.4⁎

36.5 ± 2.8⁎

42.1 ± 2.9

1

125 ± 16⁎

240 ± 65⁎

342 ± 41⁎

5

123 ± 11⁎

235 ± 44⁎

312 ± 12⁎

10

116 ± 19⁎

228 ± 38⁎

300 ± 21⁎

RBCs, Red blood cells; WBCs, white blood cells. ⁎ Significant (P b 0.05, all groups compared with control). † All parameters were expressed as mean values ± SD (n = 6).

leading to reduction in size. As we increased the amount of cross-linker more groups were cross-linked and subsequently caused a higher degree of reticulation, as suggested by Leo et al 14 In contrast, the PI was found to be minimum at 200 μL of glutaraldehyde. The probable reason could be that this amount was sufficient to cause uniform reduction in particle size for the number of GNPs formed. The deviation to either higher or lower amounts may have caused disproportionate availability of crosslinker for the same number of GNPs formed, leading to a higher PI. The EEs were not significantly affected by the amount of glutaraldehyde used (P N .05). However, type A gelatin showed approximately double the EE of type B, which might be due to formation of the larger size particles leading to smaller surface area and consequently low EE. Thus, a 200-μL concentration yielding GNPs with the desired particle size, minimum PI, and maximum EE was considered as optimum. Similarly, temperature was one important formulation variable, which had a pronounced effect on size and PI while affecting EE to a lesser extent. A higher temperature was found to be preferable for the formation of GNPs because of the high viscosity of gelatin at room temperature. The possible reason for this effect is the triple-helical structure of gelatin, which uncoiled as temperature rose in a controlled manner. However, at higher temperatures (50° ± 1°C and 60° ± 1°C) an unexpected increase in particle size was observed, probably due to complete uncoiling of gelatin chains. The results were consistent with the earlier findings of Azarmi et al 30 Likewise, pH at the second desolvation step

was significant to obtain GNPs of desired size and low PI, because formation of GNPs probably was associated with a higher degree of electrostatic interactions causing charge neutralization and consequently formation of GNPs on adding desolvating agent to polymer solution. 33 Such pHdependent behavior of gelatin could be attributed to its polyelectrolyte nature (contains both amino and carboxylate-terminated chains at its isoelectric point: type A, pH 7-9; type B, pH 4-5), and as the pH was shifted to the acidic or basic range there was a predominance of NH3+ or COO – ions depending upon the type of gelatin. 33 Drug EE is an important index for drug delivery systems. The problem associated with AmB to load in any colloidal drug delivery system is its poor solubility in most of the solvents. For the EE study, drug was dissolved in a specified quantity of DMSO and mixed with an aqueous solution of polymer with continuous stirring in a mechanical stirrer. The entrapment of AmB in GNPs could be explained on the basis of preferential localization of drug inside the nanoparticulate core, which was less hydrophilic as compared with the outer aqueous environment; moreover, as desolvation removed water from the core, EE for this drug was further improved. Low entrapment of AmB in type B gelatin GNPs could be further explained by the low surface-to-volume ratio for the number of nanoparticles formed, which could be correlated with the greater size of GNPs formed. Although gelatin and drug showed pH-dependent charge interactions evident by significant change in zeta potential values (P b .05), these charge interactions do not contribute to AmB loading in GNPs;

M. Nahar et al / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 252–261

Figure 4. Nephrotoxicity study of GNPA300 on post-injection effects at day 8 after seven doses (1 mg/kg, 5 mg/kg, and 10 mg/kg per day of AmB). Concentrations of (A) serum BUN and (B) serum creatinine were measured (n = 6). BUN and serum creatinine levels of plain AmB at all doses are significant in comparison with control (P b .001). BUN concentration in animals receiving formulation of AmB-loaded GNPs in a dose of 10 mg/kg is significant in comparison with control (P b .05).

this is contrary to the finding of Vandervoort and Ludwig 34 suggesting a charge-based interaction between pilocarpine and gelatin leading to increased EE. This low and average yield obtained in GNPs could be ascribed to discarding of some low-molecular-weight gelatin chains during the first desolvation step. The surface charge attribute of these GNPs could be suggested by their corresponding zeta potential. The positive charge on the surface of type A gelatin GNPs could be attributed to predominance of NH3+ groups, whereas the negative charge on the surface of type B gelatin GNPs could be attributed to a predominance of COO –, acquired during the formulation of GNPs in acidic pH (pH 3) and basic pH (pH 11), respectively. The relatively high zeta potential of GNPA300 over GNPA175 could be explained by the higher molecular weight of the former and hence higher density of amine groups at the surface. The loading of AmB significantly increased the net positive or negative charge (P b .05) in both types of GNPs. Type A gelatin and AmB were positively charged at pH 3, leading to an increase in net positive charge on loading, whereas type B gelatin and AmB were negatively charged at pH 11, leading to an increase in net negative charge. Moreover, zeta potential is an important

259

index for the stability of the GNPs suspension. A high absolute value of zeta potential indicates high electric charge on the surface of the drug-loaded GNPs, which can cause strong repellent forces among particles to prevent aggregation of the GNPs in buffer solution. The relatively high positive zeta potential value favors the high stability of formulation GNPA300 over GNPA175. UV-vis spectral analysis is very useful for deducing aggregation of AmB. UV-vis spectral data led us to deduce that AmB was present in GNPs in its monomeric form, and the slight decrease in intensities might be due to a small amount of available AmB. The AmB has been reported to cause a change in the UV-vis spectrum, with a very broad peak appearing around 329 nm with decreased intensities at 405, 364, and 382 nm, when the monomeric state is converted to the aggregate state. 11 Membrane diffusion techniques are the most widely used experimental methods for the study of the in vitro release profile of drugs incorporated in GNPs. The possible reason for the significantly higher release rate in GNPA300 is its higher bloom number, which corresponds to a higher molecular weight of the gelatin chain, leading to small particle formation, providing maximum surface area for interaction with dissolution media. The mechanistic insight of drug release may be assumed, in that GNPs possess greater hydrophilicity, leading to a greater tendency for the release medium to penetrate the particle core to cause swelling. The release rate of the drug and its appearance in the dissolution medium was governed by the matrix erosion in aqueous environment in the dialysis bag and by diffusion of drug across the membrane as well. Hemolysis is one of the major types of toxicity associated with AmB hampering its clinical manifestation. The possible reason for such toxicity is self-association of drug. Therefore, assessment of hemolytic toxicity seems to be a prerequisite while developing any formulation of AmB. The low hemolysis in all GNPs might be attributed to encapsulation of a nonaggregate form of AmB. During formulation AmB was added in an aqueous polymer phase whose pH was previously adjusted to either acidic or basic (pH 3 or 11) range (far from the pKa of the drug), thus precluding its self-association or aggregation and hence causing negligible hemolysis. The hemolytic profile of blank GNPs was also determined to observe the effect of polymer alone. There was negligible hemolysis in all blank GNPs, suggesting that polymer was not responsible for hemolysis and that any hemolysis observed was due to AmB. AmB have been reported to cause disturbance in many blood biochemical parameters like counts of RBCs, WBCs, leukocytes, and platelets, as well as hematocrit percentage and hemoglobin concentration. It is therefore necessary to evaluate the effect of GNP formulations on these biochemical indicators in an animal model. All results were compared with a control group of mice that

260

M. Nahar et al / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 252–261

received no formulation. GNP formulation was compared with a marketed liposome formulation (AmBisome) to establish its market potential. One group of animals received blank GNP formulations to establish whether the disturbance in these parameters was due to gelatin or AmB. At day 8 animals were anesthetized with ether and bled from the retro-orbital plexus. A significant decrease in platelet count was observed in all formulations as compared with control, but this decrease could not be explained. Different gelatins with various bloom numbers were used and evaluated for GNPs preparation, and type A gelatin with bloom number 300 was found to be suitable for such preparation because of its nanometric size, low PI, good EE, and low hemolytic profile. Further detailed hematological profiling of AmB-loaded GNPA300 proved its potential as a safe delivery system. Also, there was no significant increase in either BUN or serum creatinine levels up to day 7 of GNPA300 administration, thus advocating the safety of the proposed system for indications like visceral leishmaniasis or some systemic fungal infections. The drug release characteristics exhibited controlled delivery of AmB for longer duration, which may certainly improve patient compliance. Moreover, formulation and drug loading in GNPs were carried out at pH values far from the pKa of the drug. This is a major advance reported in this work to reduce the self-aggregation of AmB. It can be concluded that GNPs may be a viable, effective, and cheap alternative to other carriers for the controlled and safe delivery of AmB. We present a cost-effective drug delivery system for AmB that is easy to prepare and has potential for further development. Acknowledgment Manoj Nahar expresses his gratitude to SAIF, Punjab University, Chandigarh, India, for providing TEM facilities. References 1. Gallis HA, Drew RH, Picard WW. Amphotericin B: 30 years of clinical experience. Rev Infect Dis 1990;12:308-29. 2. Kleinberg M. What is the current and future status of conventional amphotericin B? Int J Antimicrob Agents 2006;27S:S12-6. 3. Boswell GW, Buell D, Bekersky I. AmBisome (liposomal Amphotericin B): a comparative review. J Clin Pharmacol 1998; 38:583-92. 4. Lincopan N, Mamizuka E, Carmona-Ribeiro A. Low nephrotoxicity of an effective amphotericin B formulation with cationic bilayer fragments. J Antimicrob Chemother 2005;55:727-34. 5. Vieira DB, Carmona-Ribeiro AM. Synthetic bilayer fragments for solubilization of amphotericin B. J Colloid Interface Sci 2001;244: 427-31. 6. Nahar M, Dutta T, Murugesan S, Asthana A, Mishra D, Rajkumar V, et al. Functional polymeric nanoparticles: an efficient and promising tool for active delivery of bioactives. Crit Rev Ther Drug Carrier Syst 2006;23:259-318.

7. Venier-Julienne M, Benoit J. Preparation, purification and morphology of polymeric nanoparticles as drug carriers. Pharm Acta Helv 1996;71: 121-8. 8. Espuelas MS, Legrand P, Campanero M, Appel M, Cheron M, Gamazo C, et al. Polymeric carriers for amphotericin B: in vitro activity, toxicity and therapeutic efficacy against systemic candidiasis in neutropenic mice. J Antimicrob Chemother 2003;52:419-27. 9. Espuelas MS, Legrand P, Irache JM, Gamazo C, Orecchioni AM, Devissaguet JPh, et al. Poly (e-caprolacton) nanospheres as an alternative way to reduce amphotericin B toxicity. Int J Pharm 1997;158: 19-27. 10. Tiyabbonchai W, Limpeanchob N. Formulation and characterization of Amphotericin B–chitosan–dextran sulfate nanoparticles. Int J Pharm 2007;329:142-9. 11. Lee CH, Singla A, Lee Y. Biomedical applications of collagen. Int J Pharm 2001;221:1Q22. 12. Narayami R, Panduranga RK. Controlled release of anticancer drug methotrexate from biodegradable gelatin microspheres. J Microencapsul 1994;11:69-77. 13. Cascone MG, Lazzeri L, Carmignani C, Zhu Z. Gelatin nanoparticles produced by a simple W/O emulsion as delivery system for methotrexate. J Mater Sci Mater Med 2002;13:523-6. 14. Leo E, Vandelli MA, Cameroni R, Forni F. Doxorubicin-loaded gelatin nanoparticles stabilized by glutaraldehyde: involvement of the drug in the cross-linking process. Int J Pharm 1997;12:75-82. 15. Verma AK, Sachin K, Saxena A, Bohidar HB. Release kinetics from bio-polymeric nanoparticles encapsulating protein synthesis inhibitor– Cycloheximide, for possible therapeutic applications. Curr Pharm Biotechnol 2005;6:121-30. 16. Yeh TK, Lu Z, Wientjes MG, Au JLS. Formulating paclitaxel in nanoparticles alters its disposition. Pharm Res 2005;22:867-74. 17. Bajpai AK, Choubey J. Design of gelatin nanoparticles as swelling controlled delivery system for chloroquine phosphate. J Mater Sci Mater Med 2006;17:345-58. 18. Sham JO, Zhang Y, Finlay WH, Roa WH, Lobenberg R. Formulation and characterization of spray-dried powders containing nanoparticles for aerosol delivery to the lung. Int J Pharm 2004;28: 457-67. 19. Truong-Le VL, Walsh SM, Schweibert E, Mao HQ, Guggino WB, August JT, et al. Gene transfer by DNA-gelatin nanospheres. Arch Biochem Biophys 1999;361:47-56. 20. Kaul G, Amiji M. Cellular interactions and in vitro DNA transfection studies with poly(ethylene glycol)–modified gelatin nanoparticles. J Pharm Sci 2005;94:184-98. 21. Kaul G, Amiji M. Tumor-targeted gene delivery using poly(ethylene glycol)–modified gelatin nanoparticles: in vitro and in vivo studies. Pharm Res 2005;22:951-61. 22. Balthasar S, Michaelis K, Dinauer N, von Briesen H, Kreuter J, Langer K. Preparation and characterisation of antibody modified gelatin nanoparticles as drug carrier system for uptake in lymphocytes. Biomaterials 2005;26:2723-32. 23. Dinauer N, Balthasar S, Weber C, Kreuter J, Langer K, von Briesen H. Selective targeting of antibody-conjugated nanoparticles to leukemic cells and primary T-lymphocytes. Biomaterials 2005;26: 5898-906. 24. Li JK, Wang N, Wu XS. Gelatin nanoencapsulation of protein/peptide drugs using an emulsifier-free emulsion method. J Microencapsul 1998; 15:163-72. 25. Coester CJ, Langer K, Van Briesen H, Kreuter J. Gelatin nanoparticles by two-step desolvation—a new preparation method, surface modifications and cell uptake. J Microencapsul 2000;17:187-93. 26. Leo E, Cameroni R, Forni F. Dynamic dialysis for the drug release evaluation from doxorubicin-gelatin nanoparticles conjugates. Int J Pharm 1999;180:23-30. 27. Echevarrya I, Barturen C, Renedo MJ, Dios-Vieitez MC. Highperformance liquid chromatographic determination of amphotericin B

M. Nahar et al / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 252–261 in plasma and tissue application to pharmacokinetic and tissue distribution studies in rats. J Chromatogr A 1998;819:171-6. 28. Singhai AK, Jain S, Jain NK. Evaluation of an aqueous injection of ketoprofen. Die Pharmazie 1997;52:149-54. 29. Marcondes MC, Borelli P, Yoshida N, Russo M. Acute Trypanosoma cruzi infection is associated with anemia, thrombocytopenia, leukopenia, and bone marrow hypoplasia: reversal by nifurtimox treatment. Microbes Infect 2000;2:347-52. 30. Azarmi S, Huang Y, Chen H, McQuarrie S, Abrams D, Roa W, et al. Optimization of a two-step desolvation method for preparing gelatin nanoparticles and cell uptake studies in 143B osteosarcoma cancer cells. J Pharm Pharmaceut Sci 2006;9: 124-32.

261

31. Mazerski J, Gryzbowska J, Borowski E. Influence of net charge on the aggregation and solubility on aggregation and solubility behaviour of amphotericin B and its derivatives in aqueous media. Eur Biophys J 1990;18:1-6. 32. Legrand P, Romero EA, Cohen BE, Bolard J. Effect of aggregation and solvent on the toxicity of amphotericin B to human erythrocytes. Antimicrob Agents Chemother 1992;36:2518-22. 33. Saxena A, Sachin K, Bohidar HB, Verma AK. Effect of molecular weight heterogeneity on drug encapsulation efficiency of gelatin nanoparticles. Coll Surf B: Biointerfaces 2005;45:42-8. 34. Vandervoort J, Ludwig A. Preparation and evaluation of drug loaded gelatin nanoparticles for topical ophthalmic use. Eur J Pharm Biopharm 2004;57:251-61.