Andrei Maksimenko

Versatile and Efficient Targeting Using a Single Nanoparticulate Platform: Application to Cancer and Alzheimer's Disease

A versatile and efficient functionalization strategy for polymeric nanoparticles (NPs) has been reported and successfully applied to PEGylated, biodegradable poly(alkyl cyanoacrylate) (PACA) nanocarriers. The relevance of this platform was demonstrated in both the fields of cancer and Alzheimers disease (AD). Prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC) and subsequent self-assembly in aqueous solution of amphiphilic copolymers, the resulting functionalized polymeric NPs exhibited requisite characteristics for drug delivery purposes: (i) a biodegradable core made of poly(alkyl cyanoacrylate), (ii) a hydrophilic poly(ethylene glycol) (PEG) outer shell leading to colloidal stabilization, (iii) fluorescent properties provided by the covalent linkage of a rhodamine B-based dye to the polymer backbone, and (iv) surface functionalization with biologically active ligands that enabled specific targeting. The construction method is very versatile and was illustrated by the coupling of a small library of ligands (e.g., biotin, curcumin derivatives, and antibody), resulting in high affinity toward (i) murine lung carcinoma (M109) and human breast cancer (MCF7) cell lines, even in a coculture environment with healthy cells and (ii) the β-amyloid peptide 1-42 (Aβ(1-42)), believed to be the most representative and toxic species in AD, both under its monomeric and fibrillar forms. In the case of AD, the ligand-functionalized NPs exhibited higher affinity toward Aβ(1-42) species comparatively to other kinds of colloidal systems and led to significant aggregation inhibition and toxicity rescue of Aβ(1-42) at low molar ratios.

Efficacy of dendrimer‐mediated angiostatin and TIMP‐2 gene delivery on inhibition of tumor growth and angiogenesis: In vitro and in vivo studies

Gene transfer is an attractive approach to fight cancer by targeting cancer cells or their vasculature. Our study reports the inhibition of tumor growth and angiogenesis by a nonviral method using dendrimers associated with 36‐mer anionic oligomers (ON36) for delivering angiostatin (Kringle 1–3) and tissue inhibitor of metalloproteinase (TIMP)‐2 genes. The optimal concentrations of dendrimers and ON36 for an efficient green fluorescent protein (GFP) plasmid delivery in endothelial cells (HMEC‐1) and cancer cells (MDA‐MB‐435) were first chosen. Then the efficacy of transfection was determined by testing angiostatin and TIMP‐2 secretion by Western blot and the biologic effects were evaluated. Angiostatin gene transfer markedly reduced in vitro (i) HMEC‐1 but not MDA‐MB‐435 proliferation; (ii) HMEC‐1 and MDA‐MB‐435 wound healing reparation; and (iii) capillary tube formation. TIMP‐2 gene transfer did not affect cell proliferation but strongly inhibited (i) wound healing of HMEC‐1 and MDA‐MB‐435 cells; and (ii) capillary tube formation. Supernatants of transfected‐MDA‐MB‐435 cells also inhibited the formation of angiogenic networks on Matrigel, indicating a paracrine effect. In vivo, intratumoral angiostatin or TIMP‐2 gene delivery using dendrimers associated with ON36 effectively inhibited tumor growth by 71% and 84%, respectively. Combined gene transfer resulted in 96% inhibition of tumor growth. Tumor‐associated vascularization was also greatly reduced. These findings provide a basis for the further development of nonviral delivery of genes to fight cancer.

Nanoparticles with in vivo anticancer activity from polymer prodrug amphiphiles prepared by living radical polymerization.

impor-tant limitations, which may explain the lower number ofsuccessful in vivo studies, still remain. The “burst release”, inwhich a large fraction of chemotherapeutic agent is quicklyreleased post-administration, can be harmful to patients.Poorly soluble drugs exhibit a high tendencyto crystallizationupon encapsulation. Finally, maximum achievable drug load-ings are generally only a few percent, thus the use of a largeamount of nanocarrier is required and this can lead toprohibitive toxicity in vivo.To overcome these obstacles, inspiration can be takenfrom the prodrug approach, whereby the drug is covalentlylinked to a (macro)molecule. The inactive prodrug is metab-olized in vivo into an active metabolite.

Polyisoprenoyl gemcitabine conjugates self assemble as nanoparticles, useful for cancer therapy

A series of new polyisoprenoyl prodrugs of gemcitabine, which can be formulated as nanoassemblies are described. These prodrugs were designed to improve gemcitabine efficacy and to overcome the limitations due to the systemic toxicity of this anticancer compound. In vitro biological assessment showed that these polyisoprenoyl gemcitabine nanoassemblies displayed notable cytotoxicity on several cancer cell lines, including murine melanoma cell line B16F10, human pancreatic carcinoma cell line MiaPaCa-2, human lung carcinoma cell line A549 and human breast adenocarcinoma cell line MCF7. Interestingly, it was observed that the anticancer efficacy of these nanoassemblies was dependant on the size of polyisoprenoyl moiety. The polyisoprenoyl prodrug of gemcitabine containing three isoprene units (2d) was the more active on all the cancer cell lines tested. The antitumor efficacy of the nanoassemblies (NAs) constructed with the most active prodrug 2d was further evaluated on a human pancreatic (MiaPaCa-2) carcinoma xenograft model in mice. The prodrug 2d NAs showed an increased antitumor efficacy as compared to free gemcitabine or to squalene-gemcitabine (SQ-gem, 2a) nanoassemblies. Interestingly, MiaPaCa-2 tumors that did not respond to gemcitabine were inhibited by 76% after treatment with prodrug 2d NAs, whereas SQ-gem-treated MiaPaCa-2 tumor xenografts decreased only by 41% compared to saline or to gemcitabine-treated mice. Together, these findings demonstrated that the modulation of the length of nanoassemblies polyisoprenoyl moiety made tumor cells more sensitive to gemcitabine treatment without flagrant toxicity, thus providing a significant improvement in the drug therapeutic index.

Improving the Antitumor Activity of Squalenoyl‐Paclitaxel Conjugate Nanoassemblies by Manipulating the Linker between Paclitaxel and Squalene

A series of new lipid prodrugs of paclitaxel, which can be formulated as nanoassemblies, are described. These prodrugs which are designed to overcome the limitations due to the systemic toxicity and low water solubility of paclitaxel consist of a squalene chain bound to the 2-OH of paclitaxel through a 1,4-cis,cis-dienic linker. This design allows the squalene-conjugates to self-assemble as nanoparticular systems while preserving an efficient release of the free drug, thanks to the dienic spacer. The size, steric hindrance, and functional groups of the spacer have been modulated. All these prodrugs self-assemble into nanosized aggregates in aqueous solution as characterized by dynamic light scattering and transmission electron microscopy and appear stable in water for several days as determined by particle size measurement. In vitro biological assessment shows that these squalenoyl-paclitaxel nanoparticles display notable cytotoxicity on several tumor cell lines including A549 lung cell line, colon cell line HT-29, or KB 3.1 nasopharyngeal epidermoid cell line. The cis,cis-squalenyl-deca-5,8-dienoate prodrug show improved activity over simple 2-squalenoyl-paclitaxel prodrug highlighting the favourable effect of the dienic linker. The antitumor efficacy of the nanoassemblies constructed with the more active prodrugs has been investigated on human lung (A549) carcinoma xenograft model in mice. The prodrug bearing the cis,cis-deca-5,8-dienoyl linker shows comparable antitumor efficacy to the parent drug, but reveals a much lower subacute toxicity as seen in body weight loss. Thus, nanoparticles with the incorporated squalenoyl paclitaxel prodrug may prove useful for replacement of the toxic Cremophor EL.

Gemcitabine-based therapy for pancreatic cancer using the squalenoyl nucleoside monophosphate nanoassemblies.

Gemcitabine is currently the most effective agent against advanced pancreatic cancer. However, the major therapeutic hurdles using gemcitabine include rapid inactivation by blood deaminases and fast development of cell chemoresistance, induced by down-regulation of deoxycytidine kinase or nucleoside transporters. To overcome the above drawbacks we designed recently a novel nanomedicine strategy based on squalenoyl prodrug of 5-monophosphate gemcitabine (SQdFdC-MP). This amphiphilic conjugate self-organized in water into unilamellar vesicles with a mean diameter of 100 nm. In this study the antitumor efficacy of SQdFdC-MP nanoassemblies (NAs) on chemoresistant and chemosensitive pancreatic adenocarcinoma models have been investigated. Cell viability assays showed that SQdFdC-MP NAs displayed higher antiproliferative and cytotoxic effects, particularly in chemoresistant pancreatic tumor cells. In in vivo studies, SQdFdC-MP NAs decreased significantly the growth (∼70%) of human MiaPaCa2 xenografts, also preventing tumor cell invasion, whereas native dFdC did not display any anticancer activity when tumor growth inhibition was only 35% with SQdFdC NAs. These results correlated with a reduction of Ki-67 antigen and the induction of apoptosis mediated by caspase-3 activation in tumor cells. These findings demonstrated the feasibility of utilizing SQdFdC-MP NAs to make tumor cells more sensitive to gemcitabine and thus providing an efficient new therapeutic alternative for pancreatic adenocarcinoma.

Combined antitumoral therapy with nanoassemblies of bolaform polyisoprenoyl paclitaxel/gemcitabine prodrugs

We report here the design and synthesis of novel bolaform prodrugs in which the two headgroups are constituted of gemcitabine and/or paclitaxel anticancer drugs bound together through short polyisoprenoyl spacers. Thanks to the unique auto-assembling properties of the polyisoprenoyl linker, these bolaform prodrugs were found to self-assemble in aqueous media into 100–200 nm nanoassemblies. The synthesis of the prodrugs and the characterization of the nanoassemblies by DLS, TEM and cryoTEM are described. These gemcitabine/paclitaxel nanodevices with high drug payloads displayed improved in vitro activities on several human and murine cancer cell lines comparatively to nanoassemblies of the corresponding squalenoyl drugs alone or in combination.

Lipid-Conjugation of Endogenous Neuropeptides: Improved Biotherapy against Human Pancreatic Cancer

Neuropeptides are small neuronal signaling molecules that act as neuromodulators for a variety of neural functions including analgesia, reproduction, social behavior, learning, and memory. One of the endogenous neuropeptides—Met-Enkephalin (Met-Enk), has been shown to display an inhibitory effect on cell proliferation and differentiation. Here, a novel lipid-modification approach is shown to create a small library of neuropeptides that will allow increased bioavailability and plasma stability after systemic administration. It is demonstrated, on an experimental model of human pancreatic adenocarcinoma, that lipid conjugation of Met-Enk enhances its tumor suppression efficacy compared to its nonlipidated counterparts, both in vitro and in vivo. More strikingly, the in vivo studies show that a combination therapy with a reduced concentration of Gemcitabine has suppressed the tumor growth considerably even three weeks after the last treatment.

Synthesis and Hybridization Properties of Oligonucleotide Analogues Containing Ornithine Backbone Modified with Nucleoalanines

The oligonucleotide analogues in which the entire phosphodiester backbone is replaced by a polyamide or peptide chain attract increasing attention as potential tools in molecular biology and as gene-targeted drugs. Among such analogues containing peptide chain, namely, oligonucleopeptides (ONPs), oligomers constructed on an b-amino alanine and d-ornithine backbones demonstrated a good specific affinity to complementary nucleic acids. As a part of our studies on design and preparation of nucleopeptides we report the synthesis and hybridization properties