Droplet Microfluidics Powered Hydrogel Microparticles for Stem Cell-Mediated Biomedical Applications.

Stem cell-related therapeutic technologies have garnered significant attention of the research community for their multi-faceted applications. To promote the therapeutic effects of stem cells, the strategies for cell microencapsulation in hydrogel microparticles have been widely explored, as the hydrogel microparticles have the potential to facilitate oxygen diffusion and nutrient transport alongside their ability to promote crucial cell-cell and cell-matrix interactions. Despite their significant promise, there is an acute shortage of automated, standardized, and reproducible platforms to further stem cell-related research. Microfluidics offers an intriguing platform to produce stem cell-laden hydrogel microparticles (SCHMs) owing to its ability to manipulate the fluids at the micrometer scale as well as precisely control the structure and composition of microparticles. In this review, the typical biomaterials and crosslinking methods for microfluidic encapsulation of stem cells as well as the progress in droplet-based microfluidics for the fabrication of SCHMs are outlined. Moreover, the important biomedical applications of SCHMs are highlighted, including regenerative medicine, tissue engineering, scale-up production of stem cells, and microenvironmental simulation for fundamental cell studies. Overall, microfluidics holds tremendous potential for enabling the production of diverse hydrogel microparticles and is worthy for various stem cell-related biomedical applications.

Dual drug delivery system of RAPTA-C and paclitaxel based on fructose coated nanoparticles for metastatic cancer treatment.

Ruthenium complexes have been widely studied as potential alternatives to platinum-type anticancer drugs due to their unique medical properties such as high selectivity, strong ability to inhibit solid tumour metastasis. However, non-specific biodistribution, and weak lethality of ruthenium to cancer cells limit its use in medical application. Drug delivery systems offer the ability to integrate multiple drugs in one system, which is particularly important to enhance the chemotherapeutic efficacy and to potentially achieve a synergistic effect of both drugs. Here, we report a dual drug nanocarrier that is based on a self-assembled biodegradable block copolymer, where the ruthenium complex (RAPTA-C) is chemically attached to the polymer chain, while another drug, paclitaxel (PTX), is entrapped in the core of the micelle. The dual drug delivery system was studied via in vitro tests using MDA-MB-231 breast cancer cells and it was observed that RAPTA-C in combination with PTX significantly enhanced anti-tumour and anti-metastasis activity.

Drug-Loading Content Influences Cellular Uptake of Polymer-Coated Nanocellulose.

While the effects of nanoparticle properties such as shape and size on cellular uptake are widely studied, influences exerted by drug loading have so far been ignored. In this work, nanocellulose (NC) coated by Passerini reaction with poly(2-hydroxy ethyl acrylate) (PHEA-g-NC) was loaded with various amounts of ellipticine (EPT) by electrostatic interactions. The drug-loading content was determined by UV-vis spectroscopy to range between 1.68 and 8.07 wt %. Dynamic light scattering and small-angle neutron scattering revealed an increased dehydration of the polymer shell with increasing drug-loading content, which led to higher protein adsorption and more aggregation. The nanoparticle with the highest drug-loading content, NC-EPT8.0, displayed reduced cellular uptake in U87MG glioma cells and MRC-5 fibroblasts. This also translated into reduced toxicity in these cell lines as well as the breast cancer MCF-7 and the macrophage RAW264.7 cell lines. Additionally, the toxicity in U87MG cancer spheroids was unfavorable. The nanoparticle with the best performance was found to have intermediate drug-loading content where the cellular uptake was adequately high while each nanoparticle was able to deliver a sufficiently toxic amount into the cells. Medium drug loading did not hinder uptake into cells while maintaining sufficiently toxic drug concentrations. It was concluded that while striving for a high drug-loading content is appropriate when designing clinically relevant nanoparticles, it needs to be considered that the drug can cause changes in the physicochemical properties of the nanoparticles that might cause unfavorable effects.

Chronological and Carbohydrate-Dependent Transformation of Fatty Acids in the Larvae of Black Soldier Fly Following Food Waste Treatment.

Although black soldier fly larvae (BSFL) can convert food waste into insectile fatty acids (FAs), the chronological and diet-dependent transformation of larval FAs has yet to be determined. This study focused on the dynamics of larval FA profiles following food waste treatment and characterized factors that may drive FA composition and bioaccumulation. Larval FA matters peaked on Day 11 as 7.7 ± 0.7% of food waste dry matter, maintained stably from Day 11-19, and decreased slightly from Day 19-21. The BSFL primarily utilized waste carbohydrates for FA bioconversion (Day 0-11) and shifted to waste FAs (Day 7-17) when the carbohydrates were close to depletion. The optimal time window for larvae harvest was Days 17-19, which fulfilled both targets of waste oil removal and larval FA transformation. Larval FAs were dominated by C12:0, followed by C18:2, C18:1, and C16:0. The waste-reducing carbohydrate primarily accounted for larval FA bioaccumulation (r = -0.947, p < 0.001). The increase in diet carbohydrate ratio resulted in the elevation of larval C12:0 yield, which indicated that larval C12:0-FA was primarily biosynthesized from carbohydrates and further transformed from ≥C16 FAs. This study elucidates the bioaccumulation process of larval FAs for food waste treatment and highlights the importance of waste carbohydrates for both the composition and transformation of larval FAs.

Super-Resolution Mapping of Single Nanoparticles inside Tumor Spheroids.

Cancer spheroids have structural, functional, and physiological similarities to the tumor, and have become a low-cost in vitro model to study the physiological responses of single cells and therapeutic efficacy of drugs. However, the tiny spheroid, made of a cluster of high-density cells, is highly scattering and absorptive, which prevents light microscopy techniques to reach the depth inside spheroids with high resolution. Here, a method is reported for super-resolution mapping of single nanoparticles inside a spheroid. It first takes advantage of the self-healing property of a "nondiffractive" doughnut-shaped Bessel beam from a 980 nm diode laser as the excitation, and further employs the nonlinear response of the 800 nm emission from upconversion nanoparticles, so that both excitation and emission at the near-infrared can experience minimal loss through the spheroid. These strategies lead to the development of a new nanoscopy modality with a resolution of 37 nm, 1/26th of the excitation wavelength. This method enables mapping of single nanoparticles located 55 µm inside a spheroid, with a resolution of 98 nm. It suggests a solution to track single nanoparticles and monitor their release of drugs in 3D multicellar environments.

Sugar Concentration and Arrangement on the Surface of Glycopolymer Micelles Affect the Interaction with Cancer Cells.

Glycopolymer-coated nanoparticles have attracted significant interest over the past few years, because of their selective interaction with carbohydrate receptors found on the surface of cells. While the type of carbohydrate determines the strength of the ligand-receptor interaction, the presentation of the sugar can be highly influential as the carbohydrate needs to be accessible in order to display good binding. To shine more light on the relationship between nanoparticle structure and cell uptake, we have designed several micelles based on fructose containing block copolymers, which are selective to GLUT5 receptors found on breast cancer cell lines. The polymers were based on poly-d,l-lactide (PLA), poly(2-hydroxyethyl) acrylate (PHEA), and poly(1- O-acryloyl-ß-d-fructopyranose) (P[1- O-AFru]). A set of six micelles was synthesized based on four fructose containing micelles (PLA242- b-P[1- O-AFru]41, PLA242- b-P[1- O-AFru]179, PLA242- b-P[1- O-AFru46-c-HEA214], PLA242- b-PHEA280- b-P[1- O-AFru]41) and two neutral controls (PLA247- b-PHEA53 and PLA247- b-PHEA166). SAXS analysis revealed that longer hydrophilic polymers led to lower aggregation numbers and larger hydrophilic shells, suggesting more glycopolymer mobility. Cellular uptake studies via flow cytometry and confocal laser scanning microscopy (CLSM) confirmed that the micelles based on PLA242- b-P[1- O-AFru]179 show, by far, the highest uptake by MCF-7 and MDA-MB-231 breast cancer cell lines while the uptake of all micelles by RAW264.7 cell is negligible. The same micelle displayed was far superior in penetrating MCF-7 cancer spheroids (three-dimensional (3D) model). Taking the physicochemical characterization obtained by SAXS and the in vitro results together, it could be concluded that the glycopolymer chains on the surface of micelle must display high mobility. Moreover, a high density of fructose was found to be necessary to achieve good biological activity as lowering the epitope density led immediately to lower cellular uptake. This work showed that it is crucial to understand the micelle structure in order to maximize the biological activity of glycopolymer micelles.

Multicellular Tumor Spheroids (MCTS) as a 3D In Vitro Evaluation Tool of Nanoparticles.

Multicellular tumor spheroid models (MCTS) are often coined as 3D in vitro models that can mimic the microenvironment of tissues. MCTS have gained increasing interest in the nano-biotechnology field as they can provide easily accessible information on the performance of nanoparticles without using animal models. Considering that many countries have put restrictions on animals testing, which will only tighten in the future as seen by the recent developments in the Netherlands, 3D models will become an even more valuable tool. Here, an overview on MCTS is provided, focusing on their use in cancer research as most nanoparticles are tested in MCTS for treatment of primary tumors. Thereafter, various types of nanoparticles-from self-assembled block copolymers to inorganic nanoparticles, are discussed. A range of physicochemical parameters including the size, shape, surface chemistry, ligands attachment, stability, and stiffness are found to influence nanoparticles in MCTS. Some of these studies are complemented by animal studies confirming that lessons from MCTS can in part predict the behaviour in vivo. In summary, MCTS are suitable models to gain additional information on nanoparticles. While not being able to replace in vivo studies, they can bridge the gap between traditional 2D in vitro studies and in vivo models.

Direct Polymerization of the Arsenic Drug PENAO to Obtain Nanoparticles with High Thiol-Reactivity and Anti-Cancer Efficiency.

PENAO (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid), which is a mitochondria inhibitor that reacts with adenine nucleotide translocator (ANT), is currently being trialed in patients with solid tumors. To increase the stability of the drug, the formation of nanoparticles has been proposed. Herein, the direct synthesis of polymeric micelles based on the anticancer drug PENAO is presented. PENAO is readily available for amidation reaction to form PENAO MA (4-(N-(S-penicillaminylacetyl) amino) phenylarsonous acid methacrylamide) which undergoes RAFT (reversible addition-fragmentation chain transfer) polymerization with poly(ethylene glycol methyl ether methacrylate) as comonomer and poly(methyl methacrylate) (pMMA) as chain transfer agent, resulting in p(MMA)-b-p(PEG-co-PENAO) block copolymers with 3-15 wt % of PENAO MA. The different block copolymers self-assembled into micelle structures, varying in size and stability (Dh = 84-234 nm, cmc = 0.5-82 µg mol-1) depending on the hydrophilic to hydrophobic ratio of the polymer blocks and the amount of drug in the corona of the particle. The more stable micelle structures were investigated toward 143B human osteosarcoma cells, showing an enhanced cytotoxicity and cellular uptake compared to the free drug PENAO (IC50 (PENAO) = 2.7 ± 0.3 µM; IC50 (micelle M4) = 0.8 ± 0.02 µM). Furthermore, PENAOs arsonous acid residue remains active when incorporated into a polymer matrix and conjugates to small mono and closely spaced dithiols and is able to actively target the mitochondria, which is PENAO's main target to introduce growth inhibition in cancer cells. As a result, no cleavable linker between drug and polymer was necessary for the delivery of PENAO to osteosarcoma cells. These findings provide a rationale for in vivo studies of micelle M4 versus PENAO in an osteosarcoma animal model.

Delivery of Amonafide from Fructose-Coated Nanodiamonds by Oxime Ligation for the Treatment of Human Breast Cancer.

The introduction of a strategy toward polymer/nanodiamond hybrids with high polymer grafting density and accessible polymer structural characterization is of critical importance for nanodiamonds' surface modification and bioagent attachment for their biomedical application. Here, we report a glycopolymer/nanodiamond hybrid drug delivery system, which was prepared by grafting amonafide-conjugated glycopolymers onto the surface of nanodiamonds via oxime ligation. Poly(1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-ß-d-fructopyranose)-b-poly(3-vinylbenzaldehyde-co-methyl methacrylate), featuring pendant aldehyde groups, is prepared via RAFT polymerization. The anticancer drug amonafide is conjugated to the polymer chains via imine chemistry, resulting in acid-degradable imine linkages. The obtained amonafide-conjugated glycopolymers are subsequently grafted onto the surface of aminooxy-functionalized nanodiamonds via oxime ligation. The molecular weight of the conjugated polymers is characterized by size-exclusion chromatography (SEC), while the successful conjugation and corresponding grafting density is assessed by nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), and thermogravimetric aanalysis (TGA). Our results indicate that the mass percentage of amonafide in the polymer chains is around 17% and the surface density of polymer chains is 0.24 molecules/nm2. The prepared drug delivery system has a hydrodynamic size around 380 nm with low PDI (0.3) and can effectively deliver amonafide into breast cancer cell and significantly inhibit the cancer cell viability. In 2D cell culture models, the IC50 values of ND-Polymer-AMF delivery system (7.19 µM for MCF-7; 4.92 µM for MDA-MB-231) are lower than those of free amonafide (11.23 µM for MCF-7; 13.98 µM for MDA-MB-231). An inhibited cell viability of nanodiamonds/polymer delivery system is also observed in 3D spheroids' models, suggesting that polymer-diamonds hybrid materials can be promising platforms for breast cancer therapy.

Influencing Selectivity to Cancer Cells with Mixed Nanoparticles Prepared from Albumin-Polymer Conjugates and Block Copolymers.

Albumin-based nanoparticles are widely used to delivery anticancer drug because they promote the accumulation of drugs in tumor sites. Nanoparticles with surface immobilized albumin are widely described in literature, although mixed nanoparticles with systematically modified ratios between albumin and PEG-based material are less common. In this work, hybrid nanoparticles were prepared by coassembly of a PEG-based amphiphilic block copolymer together with a polymer-protein conjugate. Poly(oligo(ethylene glycol) methyl ether acrylate)-poly(ε-caprolactone) (POEGMEA-PCL) was prepared by a combination of ring-opening polymerization and reversible addition-fragmentation chain transfer (RAFT) polymerization, while the polymer-protein conjugate was obtained by reacting poly(ε-caprolactone) with bovine serum albumin (BSA-PCL). Co-assembly of both amphiphiles at different ratios, with and without curcumin as a drug, led to hybrid nanoparticles with various amount of albumin on the particle surface. The resulting hybrid nanoparticles were similar in size (100-120 nm), but increasing the amount of albumin on the surface led to a more-negative ζ potential. The cytotoxicity of the curcumin-loaded nanoparticles was examined on several cell lines. The curcumin-loaded nanoparticles with high amount of albumin led to high cytotoxicity against breast cancer cell lines (MDA-MB-231 and MCF-7), which coincided with high cellular uptake. However, the cytotoxicity of the curcumin-loaded nanoparticles against CHO cells and RAW264.7 cells was reduced, suggesting that albumin can facilitate selectivity toward cancer cells.

Stabilization of Paclitaxel-Conjugated Micelles by Cross-Linking with Cystamine Compromises the Antitumor Effects against Two- and Three-Dimensional Tumor Cellular Models.

Paclitaxel (PTX)-conjugated micelles provide a promising tool for the treatment of prostate cancer. Core cross-linking by incorporating a disulfide bridge is a useful approach to improving the in vivo stability of polymeric micelles. This paper aims to investigate the effects of different degrees of cross-linking on the antitumor efficacy of micelles formed by poly(ethylene glycol methyl ether acrylate)-b-poly(carboxyethyl acrylate) (POEGMEA-b-PCEA-PTX) block copolymer. Both two-dimensional (2D) and three-dimensional (3D) in vitro prostate tumor cell models were used to evaluate the un-cross-linked and cross-linked micelles. The cytotoxicity decreased with an increase in the degree of cross-linking upon being tested with 2D cultured cells, and all micelles remained less cytotoxic than free PTX. In the 3D prostate MCTS model, however, there was no statistical difference between the performance of un-cross-linked micelles and free PTX, while increasing cross-linking densities led to significantly relevant decreases in the antitumor efficacy of micelles. These results are contradictory to our previous research using an irreversible cross-linker (1,8-diaminooctane) to stabilize POEGMEA-b-PCEA-PTX conjugate micelles where it was shown that cross-linking accelerates and improves the effects of the micelles when compared to those of un-cross-linked micelles. Further studies that aim to investigate the underlying mechanisms of disulfide bonds when micelles are internalized into cells are desired.

Fructose-Coated Nanodiamonds: Promising Platforms for Treatment of Human Breast Cancer.

Well-defined carboxyl end-functionalized glycopolymer Poly(1-O-methacryloyl-2,3:4,5-di-O-isopropylidene-ß-d-fructopyranose) (Poly(1-O-MAipFru)62) has been prepared via reversible addition-fragmentation chain transfer polymerization and grafted onto the surface of amine-functionalized nanodiamonds via a simple conjugation reaction. The properties of the nanodiamond-polymer hybrid materials ND-Poly(1-O-MAFru)62 are investigated using infrared spectroscopy, thermogravimetric analysis, dynamic light scattering, and transmission electron microscopy. The dispersibility of the nanodiamonds in aqueous solutions is significantly improved after the grafting of the glycopolymer. More interestingly, the cytotoxicity of amine-functionalized nanodiamonds is significantly decreased after decoration with the glycopolymer even at a high concentration (125 µg/mL). The nanodiamonds were loaded with doxorubicin to create a bioactive drug delivery carrier. The release of doxorubicin was faster in media of pH 5 than media of pH 7.4. The nanodiamond drug delivery systems with doxorubicin are used to treat breast cancer cells in 2D and 3D models. Although the 2D cell culture results indicate that all nanodiamonds-doxorubicin complexes are significantly less toxic than free doxorubicin, the glycopolymer-coated nanodiamonds-doxorubicin show higher cytotoxicity than free doxorubicin in the 3D spheroids after treatment for 8 days. The enhanced cytotoxicity of Poly(1-O-MAFru)62-ND-Dox in 3D spheroids may result from the sustained drug release and deep penetration of these nanocarriers, which play a role as a "Trojan Horse". The massive cell death after 8-day incubation with Poly(1-O-MAFru)62-ND-Dox demonstrates that glycopolymer-coated nanodiamonds can be promising platforms for breast cancer therapy.

PEGylated Albumin-Based Polyion Complex Micelles for Protein Delivery.

An increasing amount of therapeutic agents are based on proteins. However, proteins as drug have intrinsic problems such as their low hydrolytic stability. Delivery of proteins using nanoparticles has increasingly been the focus of interest with polyion complex micelles, prepared from charged block copolymer and the oppositely charged protein, as an example of an attractive carrier for proteins. Inspired by this approach, a more biocompatible pathway has been developed here, which replaces the charged synthetic polymer with an abundant protein, such as albumin. Although bovine serum albumin (BSA) was observed to form complexes with positively charged proteins directly, the resulting protein nanoparticle were not stable and aggregated to large precipitates over the course of a day. Therefore, maleimide functionalized poly(oligo (ethylene glycol) methyl ether methacrylate) (MI-POEGMEMA) (Mn = 26000 g/mol) was synthesized to generate a polymer-albumin conjugate, which was able to condense positively charged proteins, here lysozyme (Lyz) as a model. The PEGylated albumin polyion complex micelle with lysozyme led to nanoparticles between 15 and 25 nm in size depending on the BSA to Lyz ratio. The activity of the encapsulated protein was tested using Sprouty 1 (C-12; Spry1) proteins, which can act as an endogenous angiogenesis inhibitor. Condensation of Spry1 with the PEGylated albumin could improve the anticancer efficacy of Spry1 against the breast cancer cells lowering the IC50 value of the protein. Furthermore, the high anticancer efficacy of the POEGMEMA-BSA/Spry1 complex micelle was verified by effectively inhibiting the growth of three-dimensional MCF-7 multicellular tumor spheroids. The PEGylated albumin complex micelle has great potential as a drug delivery vehicle for a new generation of cancer pharmaceuticals.

Profluorescent PPV-Based Micellar System as a Versatile Probe for Bioimaging and Drug Delivery.

Although micelles are commonly used for drug delivery purposes, their long-term fate is often unknown due to photobleaching of the fluorescent labels or the use of toxic materials. Here, we present a metal-free, nontoxic, nonbleaching, fluorescent micelle that can address these shortcomings. A simple, yet versatile, profluorescent micellar system, built from amphiphilic poly(p-phenylenevinylene) (PPV) block copolymers, for use in drug delivery applications is introduced. Polymer micelles made from PPV show excellent stability for up to 1 year and are successfully loaded with anticancer drugs (curcumin or doxorubicin) without requiring introduction of physical or chemical cross-links. The micelles are taken up efficiently by the cells, which triggers disassembly, releasing the encapsulated material. Disassembly of the micelles and drug release is conveniently monitored as fluorescence of the single polymer chains appear, which enables not only to monitor the release of the payload, but in principle also the fate of the polymer over longer periods of time.

PEG Grafted-Nanodiamonds for the Delivery of Gemcitabine.

Carboxyl end-functionalized poly[poly(ethylene glycol) methyl ether methacrylate] [P(PEGMEMA)] and its block copolymer with gemcitabine substituted poly(N-hydroxysuccinimide methacrylate) [PGem-block-P(PEGMEMA)] are synthesized via reversible addition-fragmentation transfer (RAFT) polymerization. Then, two polymers are grafted onto the surface of amine-functionalized nanodiamonds to obtain [P(PEGMEMA)]-grafted nanodiamonds (ND-PEG) and [PGem-block-P(PEGMEMA)]-grafted nanodiamonds (ND-PF). Gemcitabine is physically absorbed to ND-PEG to produce ND-PEG (Gem). Two polymer-grafted nanodiamonds (i.e., with physically absorbed gemcitabine ND-PEG (Gem) and with chemically conjugated gemcitabine ND-PF) are characterized using attenuated total reflectance infrared spectroscopy, dynamic light scattering, and thermogravimetric analysis. The drug release, cytotoxicity (to seed human pancreatic carcinoma AsPC-1 cells), and cellular uptake of ND-PEG (Gem) and ND-PF are also investigated.

Core-Cross-Linking Accelerates Antitumor Activities of Paclitaxel-Conjugate Micelles to Prostate Multicellular Tumor Spheroids: A Comparison of 2D and 3D Models.

The 2D monolayer cell culture model is often the first step in the prediction of the success or failure of a nanoparticle-based drug delivery system. However, there is often poor translation between the 2D monolayer in vitro results and the nanoparticle-drug performance in vivo. One possible way of bridging this gap is the use of multicellular tumor spheroids (MCTSs) as an intermediate in vitro model due to its 3D structure. This paper aims to quantify and compare the results obtained from traditional 2D monolayer cell cultures and 3D MCTS by studying the cytotoxic effects of free paclitaxel (PTX) and paclitaxel, which has been conjugated to a poly(ethylene glycol methyl ether acrylate)-b-poly(carboxyethyl acrylate) (POEGMEA-b-PCEA-PTX) block copolymer and self-assembled to give a micellar delivery system. The core of the micelle was cross-linked with a diamino nondegradable cross-linker to compare the effects of micelle stability on the results. Although the 2D prostate tumor cell culture results indicated that all micellar variants (IC50: 193-271 nM) were significantly less toxic than free paclitaxel (IC50: 15.2 nM), the micelles showed faster and higher cytotoxicity than free PTX in the 3D prostate MCTS. The cross-linking of micelles even showed accelerated antitumor activities to the MCTS compared with un-cross-linked micelles. The results indicate that DAO-cross-linked POEGMEA-b-PCEA-PTX conjugate micelles will be a useful nanodrug carrier for prostate cancer therapy. MCTS offers a very promising method of incorporating 3D structures into in vitro testing.

Carbohydrate-Specific Uptake of Fucosylated Polymeric Micelles by Different Cancer Cell Lines.

Inspired by upregulated levels of fucosylated proteins on the surfaces of multiple types of cancer cells, micelles carrying ß-l-fucose and ß-d-glucose were prepared. A range of block copolymers were synthesized by reacting a mixture of 2-azidoethyl ß-l-fucopyranoside (FucEtN3) and 2-azideoethyl ß-d-glucopyranoside (GlcEtN3) with poly(propargyl methacrylate)-block-poly(n-butyl acrylate) (PPMA-b-PBA) using copper-catalyzed azide-alkyne cycloaddition (CuAAC). Five block copolymers were obtained ranging from 100 mol % fucose to 100% glucose functionalization. The resulting micelles had hydrodynamic diameters of around 30 nm. In this work, we show that fucosylated micelles reveal an increased uptake by pancreatic, lung, and ovarian carcinoma cell lines, whereas the uptake by the healthy cell lines (CHO) is negligible. This finding suggests that these micelles can be used for targeted drug delivery toward cancer cells.

Drug conjugation to cyclic peptide-polymer self-assembling nanotubes.

We show for the first time how polymeric nanotubes (NTs) based on self-assembled conjugates of polymers and cyclic peptides can be used as an efficient drug carrier. RAPTA-C, a ruthenium-based anticancer drug, was conjugated to a statistical co-polymer based on poly(2-hydroxyethyl acrylate) (pHEA) and poly(2-chloroethyl methacrylate) (pCEMA), which formed the shell of the NTs. Self-assembly into nanotubes (length 200-500 nm) led to structures exhibiting high activity against cancer cells.

Polyion complex micelle based on albumin-polymer conjugates: multifunctional oligonucleotide transfection vectors for anticancer chemotherapeutics.

Novel biocompatible polyion complex micelles, containing bovine serum albumin (BSA), polymer, and oligonucleotide, were synthesized as a generation of vectors for the gene transfection. Maleimide-terminated poly((N,N-dimethyl amino) ethyl methacrylate) (PDMAEMA) was prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization and subsequently deprotected. Precise one to one albumin-PDMAEMA bioconjugates have been achieved via 1,4-addition with the free thiol group on Cys34 on the BSA protein. SDS-PAGE and GPC (water) confirmed and quantified the successful conjugation. The conjugation efficiency was found to be independent of the molecular weight of PDMAEMA. After careful pH adjustment, the conjugate could efficiently condense anticancer oligonucleotide, ISIS 5132, which resulted in particles of 15-35 nm with a negative zeta-potential. The size was easily controlled by the polymer chain length. The albumin corona provides complete protection of the cationic polymer and genetic drug, which gave rise to lower potential toxicity from the polymer and higher gene transfection efficiency. Although a control experiment with a traditional PEG-based polyion complex micelle could deliver the drug just as effectively, if not more so, to the ovarian cancer cell line OVCAR-3, this carrier had no selectivity toward cancerous cells and proved just as toxic to HS27 (fibroblast) cell line. In contrast, the albumin-coated particles demonstrated desirable selectivity toward cancerous cells and have been shown to have outstanding performance in the cytotoxicity tests of several carcinoma monolayer cell models. In addition, the complex micelles were able to destroy pancreatic multicellular tumor spheroids, while free ISIS 5132 could not penetrate the spheroid at all. Hence, albumin-coated/oligonucleotide complex micelles are far more promising than the most classical gene delivery vectors.

3D modular bioceramic scaffolds for the investigation of the interaction between osteosarcoma cells and MSCs.

Recent advances in bone tissue engineering have shown promise for bone repair post osteosarcoma excision. However, conflicting research on mesenchymal stem cells (MSCs) has raised concerns about their potential to either promote or inhibit tumor cell proliferation. It is necessary to thoroughly understand the interactions between MSCs and tumor cells. Most previous studies only focused on the interactions between cells within the tumor tissues. It has been challenging to develop an in vitro model of osteosarcoma excision sites replicating the complexity of the bone microenvironment and cell distribution. In this work, we designed and fabricated modular bioceramic scaffolds to assemble into a co-culture model. Because of the bone-like composition and mechanical property, tricalcium phosphate bioceramic could mimic the bone microenvironment and recapitulate the cell-extracellular matrix interaction. Moreover, the properties for easy assembly enabled the modular units to mimic the spatial distribution of cells in the osteosarcoma excision site. Under this co-culture model, MSCs showed a noticeable tumor-stimulating effect with a potential risk of tumor recurrence. In addition, tumor cells also could inhibit the osteogenic ability of MSCs. To undermine the stimulating effects of MSCs on tumor cells, we present the methods of pre-differentiated MSCs, which had lower expression of IL-8 and higher expression of osteogenic proteins. Both in vitro and in vivo studies confirm that pre-differentiated MSCs could maintain high osteogenic capacity without promoting tumor growth, offering a promising approach for MSCs' application in bone regeneration. Overall, 3D modular scaffolds provide a valuable tool for constructing hard tissue in vitro models. STATEMENT OF SIGNIFICANCE: Bone tissue engineering using mesenchymal stem cells (MSCs) and biomaterials has shown promise for bone repair post osteosarcoma excision. However, conflicting researches on MSCs have raised concerns about their potential to either promote or inhibit tumor cell proliferation. It remains challenges to develop in vitro models to investigate cell interactions, especially of osteosarcoma with high hardness and special composition of bone tissue. In this work, modular bioceramic scaffolds were fabricated and assembled to co-culture models. The interactions between MSCs and MG-63 were manifested as tumor-stimulating and osteogenesis-inhibiting, which means potential risk of tumor recurrence. To undermine the stimulating effect, pre-differentiation method was proposed to maintain high osteogenic capacity without tumor-stimulating, offering a promising approach for MSCs' application in bone regeneration.