Evaluation of paclitaxel-loaded polymeric nanoparticles in 3D tumor model: impact of tumor stroma on penetration and efficacy.

Since tumor stroma poses as a barrier to achieve efficacy of nanomedicines, it is essential to evaluate nano-chemotherapeutics in stroma-mimicking 3D models that reliably predict their behavior regarding these hurdles limiting efficacy. In this study, we evaluated the effect of paclitaxel-loaded polymeric micelles (PTX-PMCs) and polymeric nanoparticles (PTX-PNPs) in a tumor stroma-mimicking 3D in vitro model. PTX-PMCs (77 nm) based on a amphiphilic block copolymer of mPEG-b-p(HPMAm-Bz) and PTX-PNPs (159 nm) based on poly(lactic-co-glycolic acid) were prepared, which had an encapsulation efficiency (EE%) of 81 ± 15% and 45 ± 8%, respectively. 3D homospheroids of mouse 4T1 breast cancer cells and heterospheroids of NIH3T3 fibroblasts and 4T1 (5:1 ratio) were prepared and characterized with high content two-photon microscopy and immunostaining. Data showed an induction of epithelial-mesenchymal transition (α-SMA) in both homo- and heterospheroids, while ECM (collagen) deposition only in heterospheroids. Two-photon imaging revealed that both fluorescently labeled PMCs and PNPs penetrated into the core of homospheroids and only PMCs penetrated into heterospheroids. Furthermore, PTX-PMCs, PTX-PNPs, and free PTX induced cytotoxicity in tumor cells and fibroblasts grown as monolayer, but these effects were substantially reduced in 3D models, in particular in heterospheroids. Gene expression analysis showed that heterospheroids had a significant increase of drug resistance markers (Bcl2, Abgc2) compared to 2D or 3D monocultures. Altogether, this study shows that the efficacy of nanotherapeutics is challenged by stroma-induced poor penetration and development of resistant phenotype. Therefore, this tumor stroma-mimicking 3D model can provide an excellent platform to study penetration and effects of nanotherapeutics before in vivo studies.

Apoptosis-inducing peptide loaded in PLGA nanoparticles induces anti-tumor effects in vivo.

Induction of apoptosis in tumor cells specifically within the complex tumor microenvironment is highly desirable to kill them efficiently and to enhance the effects of chemotherapy. Second mitochondria-derived activator of caspase (Smac) is a key pro-apoptotic pathway which can be activated with a Smac mimetic peptide. However, in vivo application of peptides is hampered by several limitations such as poor pharmacokinetics, rapid elimination, enzymatic degradation, and insufficient intracellular delivery. In this study, we developed a nanosystem to deliver a Smac peptide to tumor by passive targeting. We first synthesized a chimeric peptide that consists of the 8-mer Smac peptide and a 14-mer cell penetrating peptide (CPP) and then encapsulated the Smac-CPP into polymeric nanoparticles (Smac-CPP-NPs). In vitro, Smac-CPP-NPs were rapidly internalized by 4T1 mammary tumor cells and subsequently released Smac-CPP into the cells, as shown with fluorescence microscopy. Furthermore, Smac-CPP-NPs induced apoptosis in tumor cells, as confirmed with cell viability and caspase 3/7 assays. Interestingly, combination of Smac-CPP-NPs with doxorubicin (dox), a clinically used cytostatic drug, showed combined effects in vitro in 4T1 cells. The effect was significantly better than that of SMAC-CPP-NPs alone as well as empty nanoparticles and dox. In vivo, co-treatment with Smac-CPP-NPs and free dox reduced the tumor growth to 85%. Furthermore, the combination of Smac-CPP-NPs and free dox showed reduced proliferating tumor cells (Ki-67 staining) and increased apoptotic cells (cleaved caspase-3 staining) in tumors. In conclusion, the present study demonstrates that the intracellular delivery of Smac-mimetic peptide using nanoparticle system can be an interesting strategy to attenuate the tumor growth and to potentiate the therapeutic efficacy of chemotherapy in vivo.

Tumor stroma-containing 3D spheroid arrays: A tool to study nanoparticle penetration.

Nanoparticle penetration through tumor tissue after extravasation is considered as a key issue for tumor distribution and therapeutic effects. Most tumors possess abundant stroma, a fibrotic tissue composed of cancer-associated fibroblasts (CAFs) and extracellular matrix (ECM), which acts as a barrier for nanoparticle penetration. There is however a lack of suitable in vitro systems to study the tumor stroma penetration of nanoparticles. In the present study, we developed and thoroughly characterized a 3D co-culture spheroidal array to mimic tumor stroma and investigated the penetration of silica and PLGA nanoparticles in these spheroids. First, we examined human breast tumor patient biopsies to characterize the content and organization of stroma and found a high expression of alpha-smooth muscle actin (α-SMA; 40% positive area) and collagen-1 (50% positive area). Next, we prepared homospheroids of 4T1 mouse breast cancer cells or 3T3 mouse fibroblasts alone as well as heterospheroids combining 3T3 and 4T1 cells in different ratios (1:1 and 5:1) using a microwell array platform. Confocal live imaging revealed that fibroblasts distributed and reorganized within 48h in heterospheroids. Furthermore, immunohistochemical staining and gene expression analysis showed a proportional increase of α-SMA and collagen in heterospheroids with higher fibroblast ratios attaining 35% and 45% positive area at 5:1 (3T3:4T1) ratio, in a good match with the clinical breast tumor stroma. Subsequently, we studied the penetration of high and low negatively charged fluorescent silica nanoparticles (30nm; red and 100 or 70nm; green; zeta potential: -40mV and -20mV) and as well as Cy5-conjugated pegylated PLGA nanoparticles (200nm, -7mV) in both homo- and heterospheroid models. Fluorescent microscopy on spheroid cryosections after incubation with silica nanoparticles showed that 4T1 homospheroids allowed a high penetration of about 75-80% within 24h, with higher penetration in case of the 30nm nanoparticles. In contrast, spheroids with increasing fibroblast amounts significantly inhibited NP penetration. Silica nanoparticles with a less negative zeta potential exhibited lesser penetration compared to highly negative charged nanoparticles. Subsequently, similar experiments were conducted using Cy5-conjugated pegylated PLGA nanoparticles and confocal laser scanning microscopy; an increased nanoparticle penetration was found in 4T1 homospheroids until 48h, but significantly lower penetration in heterospheroids. Furthermore, we also developed human homospheroids (MDA-MB-231 or Panc-1 tumor cells) and heterospheroids (MDA-MB-231/BJ-hTert and Panc-1/pancreatic stellate cells) and performed silica nanoparticle (30 and 100nm) penetration studies. As a result, heterospheroids had significantly a lesser penetration of the nanoparticles compared to homospheroids. In conclusion, our data demonstrate that tumor stroma acts as a strong barrier for nanoparticle penetration. The 30-nm nanoparticles with low zeta potential favor deeper penetration. Furthermore, the herein proposed 3D co-culture platform that mimics the tumor stroma, is ideally suited to systematically investigate the factors influencing the penetration characteristics of newly developed nanomedicines to allow the design of nanoparticles with optimal penetration characteristics.