3D Bioprinted In Vitro Metastatic Models via Reconstruction of Tumor Microenvironments

The development of 3D in vitro models capable of recapitulating native tumor microenvironments could improve the translatability of potential anticancer drugs and treatments. Here, 3D bioprinting techniques are used to build tumor constructs via precise placement of living cells, functional biomaterials, and programmable release capsules. This enables the spatiotemporal control of signaling molecular gradients, thereby dynamically modulating cellular behaviors at a local level. Vascularized tumor models are created to mimic key steps of cancer dissemination (invasion, intravasation, and angiogenesis), based on guided migration of tumor cells and endothelial cells in the context of stromal cells and growth factors. The utility of the metastatic models for drug screening is demonstrated by evaluating the anticancer efficacy of immunotoxins. These 3D vascularized tumor tissues provide a proof‐of‐concept platform to i) fundamentally explore the molecular mechanisms of tumor progression and metastasis, and ii) preclinically identify therapeutic agents and screen anticancer drugs.     

Inhibition of 3‐D Tumor Spheroids by Timed‐Released Hydrophilic and Hydrophobic Drugs from Multilayered Polymeric Microparticles

First‐line cancer chemotherapy necessitates high parenteral dosage and repeated dosing of a combination of drugs over a prolonged period. Current commercially available chemotherapeutic agents, such as Doxil and Taxol, are only capable of delivering single drug in a bolus dose. The aim of this study is to develop dual‐drug‐loaded, multilayered microparticles and to investigate their antitumor efficacy compared with single‐drug‐loaded particles. Results show hydrophilic doxorubicin HCl (DOX) and hydrophobic paclitaxel (PTX) localized in the poly(dl ‐lactic‐co‐glycolic acid, 50:50) (PLGA) shell and in the poly(l ‐lactic acid) (PLLA) core, respectively. The introduction of poly[(1,6‐bis‐carboxyphenoxy) hexane] (PCPH) into PLGA/PLLA microparticles causes PTX to be localized in the PLLA and PCPH mid‐layers, whereas DOX is found in both the PLGA shell and core. PLGA/PLLA/PCPH microparticles with denser shells allow better control of DOX release. A delayed release of PTX is observed with the addition of PCPH. Three‐dimensional MCF‐7 spheroid studies demonstrate that controlled co‐delivery of DOX and PTX from multilayered microparticles produces a greater reduction in spheroid growth rate compared with single‐drug‐loaded particles. This study provides mechanistic insights into how distinctive structure of multilayered microparticles can be designed to modulate the release profiles of anticancer drugs, and how co‐delivery can potentially provide better antitumor response.     

Facile One Step Formation and Screening of Tumor Spheroids Using Droplet‐Microarray Platform

Tumor spheroids or microtumors are important 3D in vitro tumor models that closely resemble a tumor's in vivo “microenvironment” compared to 2D cell culture. Microtumors are widely applied in the fields of fundamental cancer research, drug discovery, and precision medicine. In precision medicine tumor spheroids derived from patient tumor cells represent a promising system for drug sensitivity and resistance testing. Established and commonly used platforms for routine screenings of cell spheroids, based on microtiter plates of 96‐ and 384‐well formats, require relatively large numbers of cells and compounds, and often lead to the formation of multiple spheroids per well. In this study, an application of the Droplet Microarray platform, based on hydrophilic–superhydrophobic patterning, in combination with the method of hanging droplet, is demonstrated for the formation of highly miniaturized single‐spheroid‐microarrays. Formation of spheroids from several commonly used cancer cell lines in 100 nL droplets starting with as few as 150 cells per spheroid within 24–48 h is demonstrated. Established methodology carries a potential to be adopted for routine workflows of high‐throughput compound screening in 3D cancer spheroids or microtumors, which is crucial for the fields of fundamental cancer research, drug discovery, and precision medicine.     

Superporous Poly(ethylene glycol) Diacrylate Cryogel with a Defined Elastic Modulus for Prostate Cancer Cell Research

The physical and mechanical properties of the tumor microenvironment are crucial for the growth, differentiation and migration of cancer cells. However, such microenvironment is not found in the geometric constraints of 2D cell culture systems used in many cancer studies. Prostate cancer research, in particular, suffers from the lack of suitable in vitro models. Here a 3D superporous scaffold is described with thick pore walls in a mechanically stable and robust architecture to support prostate tumor growth. This scaffold is generated from the cryogelation of poly(ethylene glycol) diacrylate to produce a defined elastic modulus for prostate tumor growth. Lymph node carcinoma of the prostate (LNCaP) cells show a linear growth over 21 d as multicellular tumor spheroids in such a scaffold with points of attachments to the walls of the scaffold. These LNCaP cells respond to the growth promoting effects of androgens and demonstrate a characteristic cytoplasmic‐nuclear translocation of the androgen receptor and androgen‐dependent gene expression. Compared to 2D cell culture, the expression or androgen response of prostate cancer specific genes is greatly enhanced in the LNCaP cells in this system. This scaffold is therefore a powerful tool for prostate cancer studies with unique advantages over 2D cell culture systems.     

A 3D In Vitro Cancer Model as a Platform for Nanoparticle Uptake and Imaging Investigations

In order to maximize the potential of nanoparticles (NPs) in cancer imaging and therapy, their mechanisms of interaction with host tissue need to be fully understood. NP uptake is known to be dramatically influenced by the tumor microenvironment, and an imaging platform that could replicate in vivo cellular conditions would make big strides in NP uptake studies. Here, a novel NP uptake platform consisting of a tissue‐engineered 3D in vitro cancer model (tumoroid), which mimics the microarchitecture of a solid cancer mass and stroma, is presented. As the tumoroid exhibits fundamental characteristics of solid cancer tissue and its cellular and biochemical parameters are controllable, it provides a real alternative to animal models. Furthermore, an X‐ray fluorescence imaging system is developed to demonstrate 3D imaging of GNPs and to determine uptake efficiency within the tumoroid. This platform has implications for optimizing the targeted delivery of NPs to cells to benefit cancer diagnostics and therapy.     

聚D,L-乳酸的体外可降解性能

研究了中等分子量(-Mη=1.10×105)的聚D,L-乳酸在体外不同环境中的可降解性能,包括不同pH值溶液(37℃)和自然土壤。降解性能采用失重率、分子量变化、溶液pH值变化等来进行评价,并对试样表面形貌采用扫描电镜(SEM)进行了观察。结果表明:聚D,L-乳酸在体外环境中具有良好的可降解性,受降解环境的影响较大;失重和分子量的减小并不平行;在不同pH值溶液和土壤中的分子量变化在一定时间内符合一级反应动力学,且随溶液pH值的增大,降解速率减小。     

Preparation and In Vitro/In Vivo Evaluation of Gliclazide Loaded Eudragit Nanoparticles as a Sustained Release Carriers

ABSTRACT

The aim of this study was to formulate and optimize gliclazide-loaded Eudragit nanoparticles (Eudragit L100 and Eudragit RS) as a sustained release carrier with enhanced efficacy. Eudragit L 100 nanoparticles (ELNP) were prepared by controlled precipitation method whereas Eudragit RSPO nanoparticles (ERSNP) were prepared by solvent evaporation method. The influence of various formulation factors (stirring speed, drug:polymer ratio, homogenization, and addition of surfactants) on particle size, drug loading, and encapsulation efficiency were investigated. The developed Eudragit nanoparticles (L100 and RS) showed high drug loading and encapsulation efficiencies with nanosize. Mean particle size altered by changing the drug:polymer ratio and stirring speed. Addition of surfactants showed a promise to increase drug loading, encapsulation efficiency, and decreased particle size of ELNP as well as ERSNP. Dissolution study revealed sustained release of gliclazide from Eudragit L100 as well as Eudragit RSPO NP. SEM study revealed spherical morphology of the developed Eudragit (L100 and RS) NP. FT-IR and DSC studies showed no interaction of gliclazide with polymers. Stability studies revealed that the gliclazide-loaded nanoparticles were stable at the end of 6 months. Developed Eudragit NPs revealed a decreased t_min (ELNP), and enhanced bioavailability and sustained activity (ELNP and ERSNP) and hence superior activity as compared to plain gliclazide in streptozotocin induced diabetic rat model and glucose-loaded diabetic rat model. The developed Eudragit (L100 and RSPO) NP could reduce dose frequency, decrease side effects, and improve patient compliance.     

High‐Resolution 3D NIR‐II Photoacoustic Imaging of Cerebral and Tumor Vasculatures Using Conjugated Polymer Nanoparticles as Contrast Agent

Exogenous contrast‐agent‐assisted NIR‐II optical‐resolution photoacoustic microscopy imaging (ORPAMI) holds promise to decipher wide‐field 3D biological structures with deep penetration, large signal‐to‐background ratio (SBR), and high maximum imaging depth to depth resolution ratio. Herein, NIR‐II conjugated polymer nanoparticle (CP NP) assisted ORPAMI is reported for pinpointing cerebral and tumor vasculatures. The CP NPs exhibit a large extinction coefficient of 48.1 L g^?1 at the absorption maximum of 1161 nm, with an ultrahigh PA sensitivity up to 2 µg mL^?1. 3D ORPAMI of wide‐field mice ear allows clear visualization of regular vasculatures with a resolution of 19.2 µm and an SBR of 29.3 dB at the maximal imaging depth of 539 µm. The margin of ear tumor composed of torsional dense vessels among surrounding normal regular vessels can be clearly delineated via 3D angiography. In addition, 3D whole‐cortex cerebral vasculatures with large imaging area (48 mm²), good resolution (25.4 µm), and high SBR (22.3 dB) at a depth up to 1001 µm are clearly resolved through the intact skull. These results are superior to the recently reported 3D NIR‐II fluorescence confocal vascular imaging, which opens up new opportunities for NIR‐II CP‐NP‐assisted ORPAMI in various biomedical applications.     

Two in One: Light as a Tool for 3D Printing and Erasing at the Microscale

The ability to selectively remove sections from 3D‐printed structures with high resolution remains a current challenge in 3D laser lithography. A novel photoresist is introduced to enable the additive fabrication of 3D microstructures at one wavelength and subsequent spatially controlled cleavage of the printed resist at another wavelength. The photoresist is composed of a difunctional acrylate cross‐linker containing a photolabile o‐nitrobenzyl ether moiety. 3D microstructures are written by photoinduced radical polymerization of acrylates using Ivocerin as photoinitiator upon exposure to 900 nm laser light. Subsequent scanning using a laser at 700 nm wavelength allows for the selective removal of the resist by photocleaving the o‐nitrobenzyl group. Both steps rely on two‐photon absorption. The fabricated and erased features are imaged using scanning electron microscopy (SEM) and laser scanning microscopy (LSM). In addition, a single wire bond is successfully eliminated from an array, proving the possibility of complete or partial removal of structures on demand.     

3D Printable Gelatin Methacryloyl (GelMA)-Dextran Aqueous Two-Phase System with Tunable Pores Structure and Size Enables Physiological Behavior of Embedded Cells In Vitro

The restricted porosity of most hydrogels established for in vitro 3D tissue engineering applications limits embedded cells with regard to their physiological spreading, proliferation, and migration behavior. To overcome these confines, porous hydrogels derived from aqueous two-phase systems (ATPS) are an interesting alternative. However, while developing hydrogels with trapped pores is widespread, the design of bicontinuous hydrogels is still challenging. Herein, an ATPS consisting of photo-crosslinkable gelatin methacryloyl (GelMA) and dextran is presented. The phase behavior, monophasic or biphasic, is tuned via the pH and dextran concentration. This, in turn, allows the formation of hydrogels with three distinct microstructures: homogenous nonporous, regular disconnected-pores, and bicontinuous with interconnected-pores. The pore size of the latter two hydrogels can be tuned from ≈4 to 100 µm. Cytocompatibility of the generated ATPS hydrogels is confirmed by testing the viability of stromal and tumor cells. Their distribution and growth pattern are cell-type specific but are also strongly defined by the microstructure of the hydrogel. Finally, it is demonstrated that the unique porous structure is sustained when processing the bicontinuous system by inkjet and microextrusion techniques. The proposed ATPS hydrogels hold great potential for 3D tissue engineering applications due to their unique tunable interconnected porosity.     

Development and In Vitro Evaluation of Surface Modified Poly(lactide-co-glycolide) Nanoparticles with Chitosan-4-Thiobutylamidine

ABSTRACT

The objective of this study was to develop a nanoparticulate drug delivery system based on the surface modification of poly(lactide-co-glycolide) (PLGA) nanoparticles with a thiolated chitosan. PLGA nanoparticles were prepared by the emulsification-solvent evaporation method. Immobilization of chitosan to the surface of PLGA nanoparticles via amide bonds was mediated by a carbodiimide. Thiol groups were covalently bound to the chitosan surface of particles by reaction with 2-iminothiolane. Obtained nanoparticles were characterized in vitro regarding size, zeta potential, thiol group content, stability at different pH values, mucoadhesion, and drug release. Results demonstrated that the surface modification of PLGA nanoparticles with thiolated chitosan (chitosan-TBA) leads to nanoparticles of a mean diameter of 889.5 ± 72 nm and positive zeta potential of + 24.74 mV. The modified nanoparticles contained 7.32 ± 0.24 μmol thiol groups per gram nanoparticles. The size of nanoparticles was strongly influenced by the pH of the surrounding medium, being 925.0 ± 76.3 nm at pH 2 and 577.8 ± 66.7 nm at pH 7.4. Thiolated nanoparticles showed a 3.3-fold prolonged residence time on the mucosa and an unchanged release profile in comparison to unmodified PLGA nanoparticles. These data suggest that surface modified chitosan-TBA conjugate PLGA nanoparticles have the potential to be used as mucoadhesive drug delivery system.