Biomaterials (v.33, #14)

Strategies to reduce dendritic cell activation through functional biomaterial design by Patrick S. Hume; Jing He; Kathryn Haskins; Kristi S. Anseth (3615-3625).
Dendritic cells play a key role in determining adaptive immunity, and there is growing interest in characterizing and manipulating the interactions between dendritic cells and biomaterial surfaces. Contact with several common biomaterials can induce the maturation of immature dendritic cells, but substrates that reduce dendritic cell maturation are of particular interest within the field of cell-based therapeutics where the goal is to reduce the immune response to cell-laden material carriers. In this study, we use a materials-based strategy to functionalize poly(ethylene glycol) hydrogels with immobilized immunosuppressive factors (TGF-β1 and IL-10) to reduce the maturation of immature dendritic cells. TGF-β1 and IL-10 are commonly employed as soluble factors to program dendritic cells in vitro, and we demonstrate that these proteins retain bioactivity towards dendritic cells when immobilized on hydrogel surfaces. Following stimulation with lipopolysaccharide (LPS) and/or cytokines, a dendritic cell line interacting with the surfaces of immunosuppressive hydrogels expressed reduced markers of maturation, including IL-12 and MHCII. The bioactivity of these immunomodulatory hydrogels was further confirmed with primary bone marrow-derived dendritic cells (BMDCs) isolated from non-obese diabetic (NOD) mice, as quantified by a decrease in activation markers and a significantly reduced capacity to activate T cells. Furthermore, by introducing a second signal to promote BMDC-material interactions combined with the presentation of tolerizing signals, the multifunctional PEG hydrogels were found to further increase signaling towards BMDCs, as evidenced by greater reductions in maturation markers.
Keywords: Immunomodulation; Hydrogel; Surface modification; Adhesion molecule; Photopolymerization;

In this work, we report a study of the interactions between bacteria and zwitterionic polymers using a long-range surface plasmon resonance (LR-SPR) sensor. The LR-SPR with an extended probing field allows one to perform in-situ monitoring of bacterial adhesion and to investigate bacteria–surface interactions under various conditions. Alginate-producing and -deficient Pseudomonas aeruginosa were used in order to study the role of capsular exopolysaccharide in bacterial adhesion onto a surface. Polycarboxybetaine polymers with one and two carbons in spaces between two oppositely charged moieties on polymer side chains (pCBAA-1 and pCBAA-2) were prepared in order to unveil the effects of material properties on surface resistance to bacterial adhesion. In addition, environmental factors (e.g., divalent cations) were investigated. This work demonstrates the role of polysaccharides beyond proteins in bacteria adhesion and the effective resistance of pCBAA-1 against bacterial adhesion.
Keywords: Bacterial adhesion; Long-range surface plasmon resonance; Zwitterionic polymers; Polysaccharide; Nonfouling properties;

Effect of a CCR1 receptor antagonist on systemic trafficking of MSCs and polyethylene particle-associated bone loss by Emmanuel Gibon; Zhenyu Yao; Allison J. Rao; Stefan Zwingenberger; Barbara Batke; Roberto Valladares; Robert L. Smith; Sandip Biswal; Sanjiv S. Gambhir; Stuart B. Goodman (3632-3638).
Particle-associated periprosthetic osteolysis remains a major issue in joint replacement. Ongoing bone loss resulting from wear particle-induced inflammation is accompanied by continued attempts at bone repair. Previously we showed that mesenchymal stem cells (MSCs) are recruited systemically to bone exposed to continuous infusion of ultra high molecular weight polyethylene (UHMWPE) particles. The chemokine–receptor axis that mediates this process is unknown. We tested two hypotheses: (1) the CCR1 receptor mediates the systemic recruitment of MSCs to UHMWPE particles and (2) recruited MSCs are able to differentiate into functional mature osteoblasts and decrease particle-associated bone loss. Nude mice were allocated randomly to four groups. UHMWPE particles were continuously infused into the femoral shaft using a micro-pump. Genetically modified murine wild type reporter MSCs were injected systemically via the left ventricle. Non-invasive imaging was used to assay MSC migration and bone mineral density. Bioluminescence and immunohistochemistry confirmed the chemotaxis of reporter cells and their differentiation into mature osteoblasts in the presence of infused particles. Injection of a CCR1 antagonist decreased reporter cell recruitment to the UHMWPE particle infusion site and increased osteolysis. CCR1 appears to be a critical receptor for chemotaxis of MSCs in the presence of UHMWPE particles. Interference with CCR1 exacerbates particle-induced bone loss.
Keywords: Arthroplasty; Wear debris; CCR1 receptor; Mesenchymal stem cells chemotaxis; Osteolysis;

Nanoparticles (NPs) are usually surface modified to increase endocytosis for applications in cellular imaging and gene delivery. The influence of cell culture substrates on endocytosis remains relatively unexplored. This study investigated the substrate-mediated effects on the uptake of NPs by mesenchymal stem cells (MSCs). Two types of NPs were employed, negatively charged paramagnetic iron oxide (Fe3O4) NPs (∼5 nm) and bare plasmid DNA pTRE-Tight-DsRED2 (3.3 kb, ∼5 nm), each of which were poorly endocytosed by the adipose-derived MSCs grown on tissue culture polystyrene (TCPS). When cells were cultured on chitosan or hyaluronan-modified chitosan (chitosan-HA) membranes, significant increases (>5-fold) in the intracellular uptake of Fe3O4 NPs as well as transfectability of plasmid DNA were demonstrated. The enhancement in transgene expression was more pronounced than that using the transfection agent. The beneficial effects were not caused by elevated proliferation or a change in the differentiation state of interacting MSCs. On chitosan and chitosan-HA, cells moved fast and formed spheroids. The cytoskeletal arrangement associated with the up-regulated RhoA activity during spheroid formation may have accounted for the increased endocytosis. Using different inhibitors, the endocytosis pathways were further clarified. Both Fe3O4 NPs and plasmid DNA were taken up primarily by clathrin-mediated endocytosis on chitosan (∼50%). The caveolae-mediated endocytosis on chitosan-HA was more evident (∼30–40%) than that on chitosan (<25%). For plasmid DNA but not Fe3O4 NPs, macropinocytosis also occurred on both substrates. Chitosan and chitosan-HA as cell culture substrates may activate different endocytic pathways of MSCs to increase NP internalization or plasmid transfection. The substrate-mediated endocytosis described here may represent a new and potentially attractive approach to facilitate stem cell labeling or to improve gene delivery efficiency without altering cell viability and differentiation.
Keywords: Nanoparticles (NPs); Mesenchymal stem cells (MSCs); Endocytosis; Gene transfection; Chitosan;

The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation by Liliana S. Moreira Teixeira; Jeroen C.H. Leijten; Jos W.H. Wennink; Anindita G. Chatterjea; Jan Feijen; Clemens A. van Blitterswijk; Pieter J. Dijkstra; Marcel Karperien (3651-3661).
In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and anti-inflammatory cytokines, but mechanically unstable. We hypothesized that the advantages of these systems may be combined in one hydrogel, which can be easily translated into clinical settings. Platelet lysate was successfully incorporated into Dex-TA polymer solution prior to gelation. After enzymatic crosslinking, rheological and morphological evaluations were performed. Subsequently, the effect of platelet lysate on cell migration, adhesion, proliferation and multi-lineage differentiation was determined. Finally, we evaluated the integration potential of this gel onto osteoarthritis-affected cartilage. The mechanical properties and covalent attachment of Dex-TA to cartilage tissue during in situ gel formation were successfully combined with the advantages of platelet lysate, revealing the potential of this enhanced hydrogel as a cell-free approach. The addition of platelet lysate did not affect the mechanical properties and porosity of Dex-TA hydrogels. Furthermore, platelet lysate derived anabolic growth factors promoted proliferation and triggered chondrogenic differentiation of mesenchymal stromal cells.
Keywords: Hydrogel; Platelet lysate; In situ crosslinking; Chemotaxis; Cell proliferation; Cartilage tissue engineering;

Long-term changes to in vitro preserved bioengineered human trachea and their implications for decellularized tissues by Silvia Baiguera; Costantino Del Gaudio; Massimo O. Jaus; Leonardo Polizzi; Alessandro Gonfiotti; Camilla E. Comin; Alessandra Bianco; Domenico Ribatti; Doris A. Taylor; Paolo Macchiarini (3662-3672).
Bioengineered tissues created for transplant will be expected to survive and contribute to function over the lifetime of the individual. To evaluate potential intrinsic changes and degradation of the extracellular matrix of decellularized human tissue scaffolds, human decellularized tracheas were evaluated over a one year period in vitro. Human tracheas were decellularized and stored for one year in phosphate-buffered saline at 4 °C in the presence of antibiotics and anti-mycotics, and their structural, mechanical, and angiogenic properties compared to baseline values. Results showed that stored human decellularized tracheas were increasingly degraded resulting in a loss of extracellular matrix architecture – in particular of collagenous and elastic fiber structure –and decreased mechanical and angiogenic properties. The mechanical alterations of the extracellular matrix but not the deterioration and microstructure were not improved by using a natural cross-linking agent. These findings demonstrate that human decellularized tracheas, stored for one year in phosphate-buffered saline solution at 4 °C, would not meet the demands for a tissue engineering matrix and likely would not yield a suitable graft for lifelong implantation. The degradation phenomenon observed in vitro may be further enhanced in vivo, having clinical relevance for tissues that will be transplanted long-term and this should be carefully evaluated in pre-clinical settings.
Keywords: Angiogenesis; Cross-linking; Decellularized human trachea; Degradation; Long-term integrity; Mechanical properties;

The use of chitosan based hydrogel for enhancing the therapeutic benefits of adipose-derived MSCs for acute kidney injury by Jiasheng Gao; Rongfu Liu; Jie Wu; Zhiqiang Liu; Junjie Li; Jin Zhou; Tong Hao; Yan Wang; Zhiyan Du; Cuimi Duan; Changyong Wang (3673-3681).
Transplantation of mesenchymal stem cells (MSCs) has been reported a great therapeutic potential for acute kidney injury (AKI). However, the therapeutic benefits are limited due to the low retention and survival of transplanted cells within target sites. In this study, thermosensitive chitosan chloride (CSCl) hydrogel was explored as injectable scaffold for adipose-derived MSCs (ADMSCs) delivery into ischemia/reperfusion (I/R) induced acute kidney injury (AKI). Thermosensitive CSCl hydrogels with/without ADMSCs were injected into the I/R site of rat AKI models. Dihydroethidium staining was used to detect the number of ROS in vivo. In order to track ADMSCs in vivo, ADMSCs were transfected with firefly luciferase and monomeric red fluorescent protein reporter genes (fluc-mrfp). The retention and survival of ADMSC were assessed using bioluminescence imaging, differentiation behaviors of ADMSCs were investigated using immunofluorescent and immunohistochemical staining. Proliferation and apoptosis of host renal cell in vivo were characterized by PCNA and TUNEL staining. Results suggested that CSCl hydrogels could improve the retention and survival of grafted ADMSCs, moreover, CSCl hydrogels could enhance the proliferation activity and reduce apoptosis of host renal cells. At 4 weeks, significant improvement of the renal function, microvessel density and tubular cell proliferation were observed in CSCl hydrogels with ADMSCs groups. Therefore, the application of thermosensitive CSCl hydrogel as scaffold for ADMSCs delivery into renal region could resolve the main obstacle of cell transplantation for acute kidney injury (AKI). Therefore, CSCl hydrogel is a potential cell carrier for treatment of AKI.
Keywords: Thermosensitive hydrogel; Chitosan; Adipose-derived mesenchymal stem cells; Acute kidney injury; Cell transplantation;

Augmented healing of critical-size calvarial defects by baculovirus-engineered MSCs that persistently express growth factors by Chin-Yu Lin; Yu-Han Chang; Chun-Yu Kao; Chia-Hsin Lu; Li-Yu Sung; Tzu-Chen Yen; Kun-Ju Lin; Yu-Chen Hu (3682-3692).
Repair of large calvarial bony defects remains clinically challenging because successful spontaneous calvarial re-ossification rarely occurs. Although bone marrow-derived mesenchymal stem cells (BMSCs) genetically engineered with baculovirus (BV) for transient expression of osteogenic/angiogenic factors hold promise for bone engineering, we hypothesized that calvarial bone healing necessitates prolonged growth factor expression. Therefore, we employed a hybrid BV vector system whereby one BV expressed FLP while the other harbored the BMP2 (or VEGF) cassette flanked by Frt sequences. Transduction of rabbit BMSCs with the FLP/Frt-based BV vector led to FLP-mediated episome formation, which not only extended the BMP2/VEGF expression beyond 28 days but augmented the BMSCs osteogenesis. After allotransplantation into rabbits, X-ray, PET/CT, μCT and histological analyses demonstrated that the sustained BMP2/VEGF expression remarkably ameliorated the angiogenesis and regeneration of critical-size (8 mm) calvarial defects, when compared with the group implanted with BMSCs transiently expressing BMP2/VEGF. The prolonged expression by BMSCs accelerated the bone remodeling and regenerated the bone through the natural intramembranous pathway, filling ≈83% of the area and ≈63% of the volume in 12 weeks. These data implicated the potential of the hybrid BV vector to engineer BMSCs for sustained BMP2/VEGF expression and the repair of critical-size calvarial defects.
Keywords: Baculovirus; Mesenchymal stem cells; Gene therapy; Calvarial bone defect; Tissue engineering; Sustained expression;

Nuclear localization signal-enhanced RNA interference of EZH2 and Oct4 in the eradication of head and neck squamous Cell carcinoma-derived cancer stem cells by Wen-Liang Lo; Yueh Chien; Guang-Yuh Chiou; Ling-Ming Tseng; Han-Shui Hsu; Yuh-Lih Chang; Kai-Hsi Lu; Chian-Shiu Chien; Mong-Lien Wang; Yi-Wei Chen; Pin-I. Huang; Fang-Wei Hu; Cheng-Chia Yu; Pen-Yuan Chu; Shih-Hwa Chiou (3693-3709).
Metastasis is the major cause of high mortality in head and neck squamous cell carcinoma (HNSCC), in which HNSCC-derived cancer stem cells (CSCs) may be involved. Several reports have coupled non-viral gene delivery with RNA interference (RNAi) to target specific genes in cancer cells. However, the delivery efficiency of RNAi is limited and remained to be improved. Moreover, the therapeutic effect of non-viral gene delivery approaches on HNSCC-derived CSCs is still uncertain. In this study, we found that EZH2 and Oct4 are upregulated in HNSCC-derived ALDH1+/CD44+ CSC-like cells. Polyurethane-short branch PEI (PU-PEI)-based administration of double-stranded DNA (dsDNA) encoding small interfering RNA (siRNA) against EZH2 and Oct4 (siEZH2/siOct4) led to partial anti-cancer capacity and mild suppression of CSC-like properties. By pre-conjugation of nuclear localization signal (NLS) to siRNA-expressing dsDNA, the anti-cancer efficacy was enhanced due to elevated nuclear delivery. Notably, the NLS-preconjugated siEZH2/siOct4 constructs remarkably repressed epithelial-mesenchymal transdifferentiation (EMT) and radioresistance in ALDH1+/CD44+ CSC-like cells, in which Wnt5A and CyclinD1 may be involved respectively. We furthermore demonstrated that this improved method was capable of reducing tumor growth and metastasis in vivo. Our findings may provide a feasible non-viral gene delivery method to eradicate HNSCC-derived CSCs and improve HNSCC therapy.
Keywords: Small interfering RNA; Head and neck squamous cell carcinoma; EZH2; Oct4; Nuclear localization signal;

Bifunctional combined Au-Fe2O3 nanoparticles for induction of cancer cell-specific apoptosis and real-time imaging by Wen Gao; Lifei Ji; Lu Li; Guanwei Cui; Kehua Xu; Ping Li; Bo Tang (3710-3718).
We demonstrate bifunctional combined Au-Fe2O3 nanoparticles (NPs) for selectively induction of apoptosis in cancer cells and real-time imaging. The as-prepared Au-Fe2O3 NPs combine the merits of both Au and γ-Fe2O3 NPs, maintaining excellent fluorescence quenching property and catalytic activity. Conjugated with αβ3 integrin-targeting peptide (RGD) and fluorescein isothiocyanate (FITC)-labeled capsase-3 recognition sequence (DEVD) on the Au surface, the resulting RGD/FITC–DEVD–Au-Fe2O3 NPs bind preferentially to integrin αβ3-rich human liver cancer cells (HepG2), sequentially initiate catalytic formation of hydroxyl radicals (•OH) and enable the real-time monitoring of•OH-induced caspase-3-dependent apoptosis in these cancer cells. Furthermore, the catalytic activity of RGD/FITC–DEVD–Au-Fe2O3 NPs is much higher than that of individual γ-Fe2O3 NPs due to the polarization effect at the Au-Fe2O3 interface. Such bifunctional Au-Fe2O3 NPs exhibit simultaneous targeting, therapeutic and imaging functions and are therefore promising for future therapeutic applications in cancer.
Keywords: Au-Fe2O3; Combined nanoparticles; Catalytic activity; Hydroxyl radical; Apoptosis; Real-time imaging;

Cancer stem cell labeling using poly(l-lysine)-modified iron oxide nanoparticles by Xueqin Wang; Fang Wei; Ajing Liu; Lei Wang; Jian-Chun Wang; Li Ren; Wenming Liu; Qin Tu; Li Li; Jinyi Wang (3719-3732).
Cell labeling using magnetic nanoparticles is an increasingly used approach in noninvasive behavior tracking, in vitro separation of cancer stem cells (CSCs), and CSC-based research in cancer therapy. However, the impact of magnetic labeling on the biological properties of targeted CSCs, such as self-renewal, proliferation, multi-differentiation, cell cycle, and apoptosis, remains elusive. The present study sought to explore the potential effects on biological behavior when CSCs are labeled with superparamagnetic iron oxide (SPIO) nanoparticles in vitro. The glioblastoma CSCs derived from U251 glioblastoma multiforme were labeled with poly(l-lysine) (PLL)-modified γ-Fe2O3 nanoparticles. The iron uptake of glioblastoma CSCs was confirmed through prussian blue staining, and was further quantified using atomic absorption spectrometry. The cellular viability of the SPIO-labeled glioblastoma CSCs was assessed using a fluorescein diacetate and propidium iodide double-staining protocol. The expressed specific markers and multi-differentiation of SPIO-labeled glioblastoma CSCs were comparatively assessed by immunocytochemistry and semi-quantitative RT-PCR. The effects of magnetic labeling on cell cycle and apoptosis rate of glioblastoma CSCs and their differentiated progenies were assayed using a flow cytometer. The results demonstrated that the cell viability and proliferation capacity of glioblastoma CSCs and their differentiated progenies were not affected by SPIO labeling compared with their unlabeled counterparts. Moreover, the magnetically labeled CSCs displayed an intact multi-differentiation potential, and could be sub-cultured to form new tumor spheres, which indicates the CSCs capacity for self-renewal. In addition, cell cycle distribution, apoptosis rate of the magnetically labeled glioblastoma CSCs, and their differentiated progenies were not impaired. Therefore, the SPIO-labeled CSCs could be a feasible approach in conducting further functional analysis of targeted CSCs.
Keywords: Cancer stem cell; Cell labeling; Iron oxide nanoparticle; Multi-differentiation; Self-renewal;

Cubic sub-20 nm NaLuF4-based upconversion nanophosphors for high-contrast bioimaging in different animal species by Tianshe Yang; Yun Sun; Qian Liu; Wei Feng; Pengyuan Yang; Fuyou Li (3733-3742).
A new upconversion luminescence (UCL) nanophosphors based on host matrix of cubic NaLuF4 with bright luminescence have been synthesized by a solvothermal method, facilitate the nanocrystals potential candidates for imaging in vivo, especially large-animals. The sub-20 nm NaLuF4 co-doped Yb3+ and Er3+ (Tm3+) showed about 10-fold stronger UCL emission than that of corresponding hexagonal NaYF4-based nanocrystals with a 20 nm diameter. Near-infrared to near-infrared (NIR-to-NIR) UCL emission of PAA-coated NaLuF4:20%Yb,1%Tm (PAA-LuTm) can penetrate >1.5 cm tissue of pork with high contrast. Based on super-strong UCL emission and deep penetration, PAA-LuTm as optical bioprobe has been demonstrated by in vivo UCL imaging of a normal black mouse, even rabbit with excellent signal-to-noise ratio. Furthermore, such cubic NaLuF4-based nanophosphor was applied in lymph node imaging of live Kunming mouse with rich white fur.
Keywords: Upconversion nanophosphors; NaLuF4; Large-animal imaging; Upconversion luminescence imaging;

A hexon-specific PEGylated adenovirus vector utilizing blood coagulation factor X by Hayato Matsui; Fuminori Sakurai; Kazufumi Katayama; Tomoko Yamaguchi; Sayuri Okamoto; Kohdai Takahira; Masashi Tachibana; Shinsaku Nakagawa; Hiroyuki Mizuguchi (3743-3755).
We previously developed a hexon-specific PEGylated adenovirus (Ad) vector by utilizing avidin-biotin interaction. However, the Ad vector was aggregated due to the multiple interactions between avidin and biotin, resulting in a reduction in the transduction efficiencies in the organs following systemic administration. In this study, we developed a new method for hexon-specific PEGylation by mixing Ad vectors with PEGylated blood coagulation factor X (FX) (PEG-FX). FX specifically binds to the hexon protein, suggesting that FX serves as an adaptor molecule for hexon-specific modification. Intravenous administration of the PEG-FX-associated Ad (PEG-FX-Ad) vector into conventional mice resulted in prolonged blood retention. However, the transduction efficiencies in the liver were not reduced by PEG-FX. On the other hand, in the warfarinized mice, the PEG-FX-Ad vectors exhibited a significant reduction in the liver transduction. In addition, incubation of the PEG-FX-Ad vector with unmodified FX resulted in dissociation of PEG-FX from the Ad vector, indicating that a substitution of PEG-FX with endogenous FX occurs in the blood following administration. This study demonstrates that FX can be used as an adaptor molecule for hexon-specific modification; however, modified FX might be substituted with endogenous FX in the blood.
Keywords: Adenovirus vector; Blood coagulation factor X; PEGylation; Hexon;

Stability of influenza vaccine coated onto microneedles by Hyo-Jick Choi; Dae-Goon Yoo; Brian J. Bondy; Fu-Shi Quan; Richard W. Compans; Sang-Moo Kang; Mark R. Prausnitz (3756-3769).
A microneedle patch coated with vaccine simplifies vaccination by using a patch-based delivery method and targets vaccination to the skin for superior immunogenicity compared to intramuscular injection. Previous studies of microneedles have demonstrated effective vaccination using freshly prepared microneedles, but the issue of long-term vaccine stability has received only limited attention. Here, we studied the long-term stability of microneedles coated with whole inactivated influenza vaccine guided by the hypothesis that crystallization and phase separation of the microneedle coating matrix damages influenza vaccine coated onto microneedles. In vitro studies showed that the vaccine lost stability as measured by hemagglutination activity in proportion to the degree of coating matrix crystallization and phase separation. Transmission electron microscopy similarly showed damaged morphology of the inactivated virus vaccine associated with crystallization. In vivo assessment of immune response and protective efficacy in mice further showed reduced vaccine immunogenicity after influenza vaccination using microneedles with crystallized or phase-separated coatings. This work shows that crystallization and phase separation of the dried coating matrix are important factors affecting long-term stability of influenza vaccine-coated microneedles.
Keywords: Coating formulation; Crystallization; Influenza vaccine; Microneedle patch; Phase separation; Vaccine stability;

Ruthenium-based complex nanocarriers for cancer therapy by Gaetano Mangiapia; Gerardino D’Errico; Luca Simeone; Carlo Irace; Aurel Radulescu; Antonio Di Pascale; Alfredo Colonna; Daniela Montesarchio; Luigi Paduano (3770-3782).
A new organometallic ruthenium complex, named AziRu, along with three amphiphilic nucleoside-based ruthenium complexes, ToThyRu, HoThyRu and DoHuRu, incorporating AziRu in their skeleton, have been synthesized, stabilized in POPC phospholipid formulations and studied for their antineoplastic activity. Self-aggregation behavior of these complexes was investigated, showing that the three synthesized AziRu derivatives able to form liposomes and, under specific conditions, elongated micelles. The formulations prepared in POPC proved to be stable for months and showed high in vitro antiproliferative activity. The here described results open new scenarios in the design of innovative transition metal-based supramolecular systems for anticancer drugs vectorization.
Keywords: Ruthenium-based complexes; Antineoplastic agents; Nanoaggregates; Anti-proliferative activity;