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Significant Tumor Growth Inhibition from Naturally Occurring Lipid-Containing Polymer Prodrug Nanoparticles Obtained by the Drug-Initiated Method Andrei Maksimenko, Duc Trung Bui, Didier Desmaêle, Patrick Couvreur, and Julien Nicolas Chem. Mater., Just Accepted Manuscript • Publication Date (Web): 10 Jun 2014 Downloaded from http://pubs.acs.org on June 10, 2014
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Significant Tumor Growth Inhibition from Naturally Occurring Lipid-Containing Polymer Prodrug Nanoparticles Obtained by the Drug-Initiated Method Andrei Maksimenko, Duc Trung Bui, Didier Desmaeሷle, Patrick Couvreur and Julien Nicolas* Institut Galien Paris-Sud, Univ Paris-Sud, UMR CNRS 8612, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, F-92296 Châtenay-Malabry cedex, France ABSTRACT: By using the ‘drug-initiated’ method under controlled/living radical polymerization conditions, well-defined polymer prodrug nanoparticles based on the anticancer drug gemcitabine and squalene, a natural lipid, demonstrated significant in vivo anticancer activity in a subcutaneous tumor xenograft model of pancreatic cancer, as shown by a ~75% decrease in the tumor growth, a strong reduction in normal vasculature, together with antiproliferative and anti-apoptotic properties. This general and simple approach may open a new area in the field of drug delivery as multiple drug/polymer combinations could be selected and other pathologies could be treated.
Drug-containing nanocarriers hold significant promise in nanomedicine for precisely delivering the drug to the diseased areas in the body while avoiding its nonspecific cell and tissue biodistribution as well as its rapid metabolization and excretion.1 So far, a plethora of different nanoparticulate systems, in terms of nature, architecture and features, have been designed for improving cancer therapy outcomes. Among them, liposomes2 and polymer nanoparticles/micelles3 represent the most investigated classes while the physical encapsulation of drugs into nanocarriers remains the main strategy. Yet, in order to overcome the main limitations encountered with physically encapsulated drugs (e.g., a limited and hardly tunable drug payload, the burst release, etc.), the concept of prodrug,4 whereby the drug is covalently linked to the nanocarriers, has been proposed for suppressing the burst release and leading to a sustained drug release.5 In order to further increase the therapeutic efficiency, drug delivery devices have also continuously gained in complexity and sophistication over the years, reflecting recent progress, notably in polymer science and materials chemistry. For instance, conferring them with targeting abilities6 and/or stimuli-responsiveness7 has become feasible and has opened new avenues in nanomedicine. However, the more functionality assigned to the nanocarriers, the more complex they are and the more difficult their pharmaceutical development will be. Therefore, there is a crucial need in nanomedicine of simple, yet efficient concepts to give the best possible chances to eventually reach the market. In this context, the ‘drug-initiated’ method appears as an appealing strategy. It consists in growing a short hydrophobic polymer chain from a hydrophilic drug under controlled/living radical conditions, resulting in amphiphilic drug-polymer conjugates able to form self-stabilized prodrug nanoparticles.5a This was illustrated by selecting gemcitabine (Gem) as the anticancer drug and polyisoprene as a model hydrophobic polymer due to its structural similarity with natural lipids.8 Gem was functionalized with an alkoxyamine initiator to conduct nitroxide-mediated polymerization (NMP)9 of isoprene. The resulting nanoparticles were nar-
rowly dispersed in the 100–160 nm range in diameter and demonstrated significant anticancer activity in vivo in tumor bearing mice. However, the robustness and the versatility of this method, and therefore its future potential in drug delivery, mainly depend on whether it is applicable to other drugs and other polymers, and also to other radical polymerization techniques. Furthermore, the use biorelevant polymers would be of paramount importance for obvious toxicity reasons.
Scheme 1. Synthesis of gemcitabine-poly(squalenyl methacrylate) (Gem-PSqMA) prodrug nanoparticles. In order to tackle these crucial pending questions, we have conceived a new class of amphiphilic polymer prodrug nanoparticles with polymer promoieties composed of multiple copies of squalene, a naturally occurring lipid.10 This was obtained by reversible addition-fragmentation chain transfer (RAFT)11 radical polymerization of squalenoyl methacrylate (SqMA) from a Gemfunctionalized trithiocarbonate RAFT agent (Scheme 1). Although preliminary results were encouraging,12 in vivo investigations, which are considered to be more reliable and relevant than in vitro assays, have not yet been conducted. Not only this would give a clear demonstration of the full potential of these novel lipid/polymer nanoparticles, but this would also offer new opportunities to this approach as it could be applicable to many other
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polymer/drug combinations, and therefore to other pathologies, simply by changing the nature of the drug. The Gem-based chain transfer agent was obtained from the coupling of 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid to the C-4 amino group of tert-butyldimethylsilyl (TBS)-protected Gem by conventional acylation via the mixed anhydride pathway.12 The RAFT polymerization of SqMA followed by the subsequent removal of TBS moieties yielded welldefined Gem-PSqMA prodrugs (Mn,SEC = 5 540 g.mol-1, Mn,NMR = 6 800 g.mol-1, Ð = 1.3), which correspond to ~14 pending squalene moieties per conjugate and a Gem loading of 4.2 wt.%, as determined by 1H NMR (Figure 1a–b). Note that the drug payload could be easily increased simply by reducing the polymer chain length, which is possible due to the controlled polymerization process employed here. The nanoparticles were obtained by nanoprecipitation and exhibited an average diameter of 97 ± 1 nm with a narrow particle size distribution of 0.133. The monodisperse nature of the nanoparticle formulation was highlighted by the homogeneous size distribution graphics obtained from DLS measurements expressed in intensity, volume and number (Figure 1c), and by Cryo-TEM analysis (Figure 1d). ζ-potential value was strongly negative (–48 ± 3 mV), thus indicating significant electrostatic stabilization of the nanoparticles.
Figure 1. (a) 1H NMR spectrum (300 MHz, CDCl3) of GemPSqMA. Insert: zoom of the 19F NMR spectrum in the -105 – -125 ppm region. (b) Size exclusion chromatogram in CHCl3 of GemPSqMA. (c) DLS data giving the average diameter in intensity, number and volume. (d) Cryo-TEM of Gem-PSqMA nanoparticles.
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The systemic toxicity of Gem-PSqMA nanoparticles was first investigated and compared to those of free Gem, PSqMA nanoparticles and saline after repeated intravenous (i.v.) injections to healthy nude mice. The injected dose of Gem-PSqMA nanoparticles was limited by the maximum reachable concentration of nanoparticles in solution (10 mg.mL-1) and the maximum volume able to be injected (200 µL), corresponding to a Gem equivalent dose of 3.4 mg.kg-1 per injection. Therefore, the following treatments and doses were administered by i.v. injections in the lateral tail vein on days 0, 4, 8, 11, 15, 18, 21 and 25: saline 0.9%, PSqMA nanoparticles (77 mg.kg-1, that is the same dose of polymer as for Gem-PSqMA), free Gem (3.4 mg.kg-1), and GemPSqMA nanoparticles (3.4 mg.kg-1, equiv. Gem). By using a 10% weight loss as a standard threshold value for animal health status, no toxicity was observed for all treatments (Figure S1, Supporting Information). We therefore used this protocol to evaluate the antitumor activity of the Gem-PSqMA nanoparticles against human pancreatic (MiaPaCa-2) carcinoma xenograft model in nude mice. Injections of MiaPaCa-2 cells were performed in the flank of athymic nude mice. After tumors had grown up to ~100 mm3, the same treatments were administered following the injection protocol previously defined from the systemic toxicity study. As shown in Figure 2ab, the tumor growth was not significantly affected by treatments with PSqMA nanoparticles or free Gem, compared to untreated mice (saline 0.9%), with a final tumor volume of about 1800–2000 mm3 at the end of the monitoring (day 48). This demonstrated the absence of anticancer activity of free Gem (mainly due to rapid deamination by deoxycytidine deaminase).13 Conversely, mice treated with Gem-PSqMA nanoparticles showed a drastic tumor growth inhibition of ~75% (p < 0.001) compared to untreated mice at day 48, leading to a final tumor volume as low as 575 mm3.
Figure 2. In vivo anticancer activity of Gem (▲), Gem-PSqMA nanoparticles (●), saline 0.9% (▼) and PSqMA nanoparticles (■) following intravenous treatment (on days 0, 4, 8, 11, 15, 18, 21 and 25) of mice bearing MiaPaCa-2 subcutaneous tumors: (a) tumor progression as function of time. The values are the mean ± SD (n = 5–6, *p < 0.001); (b) pictures showing the position of the
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implanted tumor on representative mouse at day 32 for all treatments; (c) relative body weight change as function of time. The relative body weight loss of nude mice was also monitored (Figure 2c). The Gem-treated group showed a marked decrease (~5%) during the first 20 days compared to control treatments (i.e., saline 0.9% and PSqMA nanoparticles), before it follows the same evolution thereafter and gives a body weight increase. Noteworthy is to point out that Gem-PSqMA nanoparticles did not cause any significant weight loss all along the monitoring. Therefore, the improved anticancer activity of GemPSqMA nanoparticles was not at the expense of the inherent toxicity of the anticancer drug. In order to have an in-depth evaluation of the therapeutic activity of the prodrug nanoparticles, immunohistochemical analysis of tumor biopsies was performed (Figure 3). HES staining showed enlarged cells with necrotic changes only in the case of GemPSqMA nanoparticle-treated tumor tissues (Figure 3a, line 1). The TUNEL staining of tumor biopsies sections (5 µm), which is a common method for detecting the DNA fragmentation resulting from apoptotic signaling cascades, was used to assess the induction of apoptosis by the different treatments (Figure 3a, line 2). The mean proportions of TUNEL positive cells/field (red color) for the groups treated with free Gem and the controls (saline 0.9% and PSqMA nanoparticles) were 0.9% and 0.6%, respectively, whereas Gem-PSqMA nanoparticles reached 16.8% (Figure 3b), thus proving the significant induction of apoptosis. Among the different signaling pathways of programmed cell death, caspase-3 plays a central role in the execution-phase of cell apoptosis. Immunostaining of the active form of caspase-3 protease revealed major caspase-3 activation (35.6%) exclusively for Gem-PSqMA nanoparticle-treated mice. In contrast, tumors from mice that received free Gem injections gave a mean value of only 5.1% of caspase-3 positive cells/field and those tested by the control injections were as low as ~0.5%. Additionally, GemPSqMA nanoparticles caused an important decrease (about –50%) of the MiaPaCa-2 tumor proliferative activity, comparatively to free Gem, as indicated by the reduced number of Ki-67-positive tumor cells (Figure 3a, line 3 and Figure 3b), which is a cellular marker for cell proliferation. As shown in Figure 3b, treatment with Gem-PSqMA nanoparticles exhibited mean values of 28% Ki-67 positive cells/field, which is drastically lower than values obtained with free Gem (59%), saline (55%) and PSqMA nanoparticles (57%). Finally, the antineovasculature effect of the Gem-PSqMA nanoparticles was confirmed by immunostaining of CD34 (Figure 3a, line 5 and Figure 3c). The CD34 protein is a member of a family of single-pass transmembrane sialomucin proteins that show expression in early hematopoietic and vascular-associated tissue.14 The more pronounced suppressive effect on neovasculature was clearly obtained by Gem-PSqMA nanoparticles compared to other treatments, leading of a mean value of 9.5% vessel area/field whereas free Gem led to 19%, which is in the similar range than values obtained from treatments with saline and PSqMA nanoparticles (20–22%). In summary, the Gem-PSqMA nanoparticles demonstrated a reduction in normal vasculature, together with antiproliferative and apoptotic effects.
Figure 3. Immunohistochemical staining of representative tumors from each group excised at day 32: (a) paraffin sections from tumor biopsies were submitted to hematoxylin-eosin-safranin staining (HES) for morphology study, TUNEL staining for visualization of apoptotic cells, Ki-67 staining for proliferated cells, caspase-3 staining for indication of caspase-3 positive cells, and CD34 staining for detection of the tumor vasculature; (b) quantification of the apoptotic, Ki-67 and caspase-3-positive cells in the tumor tissue sections (*p < 0.001); (c) percentage of the vessel area with reference to the total tumor area (*p < 0.001). By establishing the significant in vivo anticancer activity of these novel lipid-based Gem-PSqMA prodrug nanoparticles, we have demonstrated that the ‘drug-initiated’ method under controlled/living radical conditions, which consists in growing a short hydrophobic chain from a hydrophilic anticancer drug, is a truly robust and flexible, yet simple strategy to achieve efficient drug delivery. The simplicity of this system, solely composed of selfassembled amphiphilic drug-polymer prodrugs as the sole building blocks, without the need to additional surfactant, is indeed a crucial advantage over other approaches, especially in the context of bench-to-bedside translation. For instance, it offers valuable benefits compared to the traditional and widely exploited ‘conjugation to’ method5a (i.e., when the drug is linked to a preformed polymer scaffold): (i) the efficiency of the conjugation is nearly quantitative as all the drugs is retained at the chain ends; (ii) the purification of the conjugate is easier since only the unreacted monomer has to be removed as opposed to a preformed polymer
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and (iii) fine tuning of drug loading is achieved simply by varying the molecular weight of the polymer chain. Also, our findings could open exciting perspectives in the biomedical field as multiple drug/polymer combinations are now worth considering and other pathologies could be treated simply by selecting suitable drugs.
ASSOCIATED CONTENT Supporting Information. Experimental part, systemic toxicity evaluation of Gem-PSqMA nanoparticles, free Gem and PSqMA nanoparticles in mice. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author *
[email protected] Author Contributions The manuscript was written through contributions of all authors.
Funding Sources The research leading to these results received funding from the European Research Council under the European Community’s Seventh Framework Programme FP7/2007-2013 (Grant Agreement No. 249835). The PhD training program in France of the University of Science and Technology of Hanoi is also acknowledged for the financial support of D.T.B. CNRS and French ministry of research are also acknowledged for financial support. The authors declare no competing financial interests. ACKNOWLEDGMENT The authors thank Julie Mougin (UMR CNRS 8612) for the CryoTEM analysis, the service of the animal experimentation from the IFR141 IPSIT (Châtenay-Malabry, France), and Olivia Bawa and Dr Paule Opolon (Institut Gustave Roussy, Villejuif, France) for preparation of stained histological slides.
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