Biomacromolecules 2011, 12, 97–104
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Superior Preclinical Efficacy of Gemcitabine Developed As Chitosan Nanoparticulate System Jose´ L. Arias,† L. Harivardhan Reddy,‡,§ and Patrick Couvreur*,§ Departamento de Farmacia y Tecnologı´a Farmace´utica, Facultad de Farmacia, Universidad de Granada, 18071 Granada, Spain, and Faculte´ de Pharmacie, Universite´ Paris-Sud XI, UMR CNRS 8612, IFR 141, 92296 Chaˆtenay-Malabry Cedex, France Received September 3, 2010; Revised Manuscript Received November 15, 2010
Gemcitabine, an anticancer nucleoside analogue, undergoes rapid enzymatic degradation following intravenous injection. This necessitates the administration of a high order of doses to observe a required therapeutic response, while such high doses result in significant side effects. To improve the intravenous delivery of gemcitabine and simultaneously enhance its antitumor activity, we have investigated its incorporation into a drug nanoplatform based on the biodegradable polymer chitosan. Two gemcitabine loading methods have been investigated: (i) entrapment into the polymeric network (entrapment procedure): drug incorporation prior to the coacervation process that leads to the formation of gemcitabine-loaded chitosan (GemChit) nanoparticles; and (ii) surface deposition onto already formed chitosan nanoparticles after incubation in gemcitabine solution (adsorption procedure). The former method produced much higher gemcitabine loading values and a sustained release profile. The main factors determining the gemcitabine loading and release kinetic have also been analyzed. Following intravenous injection, the GemChit formulation displayed a significantly improved antitumor activity comparatively to free gemcitabine, which was further confirmed by histology and immunohistochemistry studies, suggesting the potential of this chitosan-based gemcitabine nanomedicine for the effective treatment of tumors.
1. Introduction Despite the long use of many multiple drug regimes to improve clinical success, treatment failure frequently occurs even in cancers that are sensitive to chemotherapy agents. The main reasons are (i) the relatively poor selectivity for target tissues, (ii) the large biodistribution and nonintended extravasation with severe side effects in sensitive nontarget tissues, (iii) the unfavorable pharmacokinetics (rapid clearance and in vivo degradation) that determines the use of high doses and imposes on patients a rigorous schedule for reaching an antitumor effect, (iV) the susceptibility to induce drug resistance, and (V) the physicochemical properties of many drugs (e.g., hydrophobicity and high electric charge) that promote the unsuccessful localization at the target site. In addition, the physiology of the tumor is also responsible for chemotherapy failure. The higher hydrostatic pressure inside the tumor may induce a pressure gradient that may hinder drug diffusion inside the tumor mass. Furthermore, the disorganized vasculature and lack of functional lymphatics in the tumors reduces the effective outflow of interstitial fluid and thereby increases the interstitial fluid pressure. This can contribute to the slowdown of the movement of drug molecules within the tumor and decrease their reach to the tumor cells that are away from blood vessels, thus, jeopardizing the drug efficacy.1-4 In such a case, nanoparticle formulations could be expected to increase the drug penetration in tumor tissue and may contribute to enhanced antitumor efficacy. * To whom correspondence should be addressed. Phone: (+33) 1 46 83 53 96. Fax: (+33) 1 46 61 93 34. E-mail:
[email protected]. † Universidad de Granada. ‡ Current address: Sanofi-aventis, 13 Quai Jules-Guesdes 94403 Vitrysur-Seine France. § Universite´ Paris-Sud XI.
Gemcitabine (2′,2′-difluoro-2′-deoxycytidine), a nucleoside analogue, is one of the more prescribed anticancer compounds. It is relatively well tolerated when used as a single agent in the treatment of a wide variety of cancers, including lung, colon, head and neck, and ovarian cancers. This chemotherapy agent has been also approved against pancreatic, nonsmall cell lung, bladder, and breast cancer. The antitumor action involves (i) the incorporation of its active triphosphate form (5′-triphosphategemcitabine) into the DNA strand, halting its elongation and causing cell death, and (ii) the ribonucleotide reductase inhibition. Despite its demonstrated anticancer activity, this molecule suffers from various drawbacks, particularly its rapid metabolism into the inactive uracil derivative following intravenous administration, which generates the need to use high doses resulting in severe side-effects. Hence, gemcitabine has a very short plasma half-life after intravenous administration ( 9, the zeta potential could approach a zero value. This behavior may be explained if we consider the charge generation mechanism at the polymer-solution interface. Chitosan nanoparticles were charged positively despite sulfate ions were used as precipitant. This indicates that only a small part of the amino groups of chitosan were neutralized during nanoparticle formation. These residual amino groups would be responsible for the positive ζ and, in addition, will be freely accessible for interaction with drugs as well. The pH dependence was strongest between pH 5 and 7, leveling off at pH values above 7. This may be correlated with the dissociation constant of chitosan (≈6).22 The contact angles (θ) of water, formamide, and diiodomethane on the pellets of dry chitosan nanoparticles were 60 ( 6°, 38 ( 4°, and 30 ( 4°, respectively. The γSLW, γS+, and γS components represent a set of physical magnitudes that were evaluated to define the nature of the polymer. It was determined that the γSLW component was 44.2 ( 1.6 mJ/m2, the γS+ component was 0.5 ( 0.1 mJ/m2, and the γS- component was 15.8 ( 4.9 mJ/m2. Hence, chitosan is basically a monopolar electron-donor material, capable of having acid-base interactions with phases of whatever polarity (γ+, γ-, or both, different from zero), although the acid-base forces do not contribute to its cohesion free energy.17 Additionally, these γS components manifest themselves in the hydrophobicity/hydrophilicity of chitosan nanoparticles: to determine whether a material can be considered hydrophobic or hydrophilic, the free energy of interaction ∆GSLS (not considering the electrostatic component) may be evaluated between the solid phases immersed in the liquid.16 This quantity can be written as follows per unit area of interacting particles: TOT ∆GSLS ) -2γSL
(3)
It was found that ∆GSLS was negative for chitosan nanoparticles (-27 ( 12 mJ/m2) and, thus, this polymer could be
[gemcitabine] (M)
gemcitabine entrapment efficiency (%)
gemcitabine loading (%)
10-4 5 × 10-4 10-3 5 × 10-3 10-2
1.6 ( 0.4 3.6 ( 0.6 5.1 ( 1.1 6.6 ( 1.2 7.8 ( 1.5
0.003 ( 0.001 0.029 ( 0.005 0.081 ( 0.017 0.535 ( 0.079 1.3 ( 0.2
considered hydrophobic: the interfacial interactions favor the attraction of the nanoparticles to each other (not considering the electrostatic component). 3.2. Surface Adsorption of Gemcitabine onto Chitosan Nanoparticles. It was observed that the entrapment efficiency (%) of gemcitabine increased significantly with the drug concentration in the incubation medium (i.e., from 1.6 ( 0.4 to 7.8 ( 1.5% for gemcitabine concentrations ranging from 10-4 M to 10-2 M; Table 2). Maximum drug loading was low (i.e., 1.3 ( 0.2%) and could be explained by the unfavorable thermodynamic interaction between the hydrophobic chitosan (see section 3.1) and the hydrophilic gemcitabine. Indeed, the logarithm of the partitioning coefficient of gemcitabine (in n-octanol/water, pH 7.4) was determined to be log Doct/water ) -1.24.23 Thus, the interaction of gemcitabine with the hydrophobic nanoparticle surface may be considered as not favorable. The electrokinetic analysis of the drug adsorption process qualitatively confirmed these findings: ue displayed an almost null trend to rise toward progressively more positive values as the concentration of gemcitabine was increased. In fact, ue rose from 2.6 ( 0.1 µm · s-1/V · cm-1 to 3.1 ( 0.2 µm · s-1/V · cm-1 when the chitosan nanoparticles suspension (i.e., 1 mg/mL) was brought in contact with gemcitabine concentrations ranging from 10-4 M to 10-2 M. The originally positive charge of the polymer is supposed to be only slightly increased by the very low adsorption of the antitumor drug. In any case, an unfavorable electrostatic interaction in the aqueous medium between the positively charged chitosan nanoparticles and gemcitabine (positively charged presumably by the protonation of the -NH2 group of the molecule) is expected, further confirming the poor adsorption of gemcitabine onto the chitosan nanoparticles. 3.3. Gemcitabine Entrapment into Chitosan Nanoparticles. 3.3.1. Influence of the Gemcitabine Concentration. Because of the previously discussed unfavorable electrostatic interaction between gemcitabine hydrochloride and chitosan, we have considered the possibility to improve gemcitabine loading by introducing the drug before the coacervation process (i.e.,
Efficacy of Gemcitabine As Chitosan Nanoparticulate
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Table 3. Influence of Chitosan and Pluronic F-68 Concentration on Gemcitabine Entrapment Efficiency (%) and Gemcitabine Loading (%) by Entrapment into the Polymeric Network (Entrapment Procedure) formulation conditions of chitosan nanoparticles gemcitabine [gemcitabine] [chitosan] [pluronic F-68] entrapment gemcitabine (M) (%, w/v) (%, w/v) efficiency (%) loading (%) 10–2 10–2 10–2 10–2 10–2 10–2
Figure 2. Gemcitabine entrapment efficiency (%; a) and gemcitabine loading (%; b) values into chitosan nanoparticles (entrapment procedure) as a function of the equilibrium drug concentration (the lines are guides to the eye).
entrapment procedure) to provoke the mechanical trapping of the drug. Furthermore, a stabilizing agent (i.e., pluronic F-68) was added to facilitate the opening of the chitosan chains and to yield a noncompact structure where the drug can be incorporated.13,24 Figure 2 shows the entrapment efficiency (%) and loading (%) of gemcitabine into the chitosan nanoparticles as a function of the equilibrium drug concentration. Compared to the surface adsorption procedure, both the entrapment efficiency and the drug loading were significantly enhanced whatever the gemcitabine concentration. As an example, when the initial concentration of gemcitabine in the adsorption/entrapment medium was 10-2 M, these parameters rose from 7.8 ( 1.5 and 1.3 ( 0.2% (for gemcitabine adsorption procedure) to 37.2 ( 3.3 and 11.2 ( 0.9% (for gemcitabine entrapment procedure) for, respectively, entrapment efficiency and drug loading. As can be observed in Figure 2, the gemcitabine concentration positively influenced the entrapment efficiency into the chitosan nanoparticles, similar to that observed with the adsorption procedure. 3.3.2. Influence of the Surfactant and Chitosan Concentration. Table 3 evaluates the influence of the quantity of pluronic F-68, and chitosan used in the formulation of the nanoparticles on gemcitabine loading (%) into chitosan. Gemcitabine loading was noticeably lower in the absence of pluronic F-68 but increased significantly and remained unaltered at and above 0.5% (w/v) pluronic F-68 within the concentration range tested. Interestingly, the yield % of GemChit nanoparticles was always >95% in all the formulations tested containing pluronic F-68, but decreased to
