Biodegradable and Inherently Fluorescent pH-Responsive Nanoparticles for Cancer Drug Delivery.

PURPOSE: The development of two novel pH-only and pH- and thermo-responsive theranostic nanoparticle (NP) formulations to deliver an anticancer drug and track the accumulation and therapeutic efficacy of the formulations through inherent fluorescence. METHODS: A pH-responsive formulation was synthesized from biodegradable photoluminescent polymer (BPLP) and sodium bicarbonate (SBC) via an emulsion technique, while a thermoresponsive BPLP copolymer (TFP) and SBC were used to synthesize a dual-stimuli responsive formulation via free radical co-polymerization. Cisplatin was employed as a model drug and encapsulated during synthesis. Size, surface charge, morphology, pH-dependent fluorescence, lower critical solution temperature (LCST; TFP NPs only), cytocompatibility and in vitro uptake, drug release kinetics and anticancer efficacy were assessed. RESULTS: While all BPLP-SBC and TFP-SBC combinations produced spherical nanoparticles of a size between 200-300 nm, optimal polymer-SBC ratios were selected for further study. Of these, the optimal BPLP-SBC formulation was found to be cytocompatible against primary Type-1 alveolar epithelial cells (AT1) up to 100 µg/mL, and demonstrated sustained drug release over 14 days, dose-dependent uptake, and marked pH-dependent A549 cancer cell killing (72 vs. 24% cell viability, at pH 7.4 vs. 6.0). The optimal TFP-SBC formulation showed excellent cytocompatibility against AT1 cells up to 500 µg/mL, sustained release characteristics, dose-dependent uptake, pH-dependent (78% at pH 7.4 vs. 64% at pH 6.0 at 37°C) and marked temperature-dependent A549 cancer cell killing (64% at 37°C vs. 37% viability at pH 6.0, 41°C). CONCLUSIONS: In all, both formulations hold promise as inherently fluorescent, stimuli-responsive theranostic platforms for passively targeted anti-cancer therapy.

Development and characterization of a novel poly(N-isopropylacrylamide)-based thermoresponsive photoink and its applications in DLP bioprinting.

The field of 3-dimensional (3D) bioprinting has significantly expanded capabilities in producing precision-engineered hydrogel constructs, and recent years have seen the development of various stimuli-responsive bio- and photoinks. There is, however, a distinct lack of digital light processing (DLP)-compatible photoinks with thermoresponsivity. To remedy this, this work focuses on formulating and optimizing a versatile ink for DLP printing of thermoresponsive hydrogels, with numerous potential applications in tissue engineering, drug delivery, and adjacent biomedical fields. Photoink optimization was carried out using a multifactorial study design. The optimized photoink yielded crosslinked hydrogels with strong variations in hydrophobicity (contact angles of 44.4° LCST), indicating marked thermoresponsivity. Mechanical- and rheological characterization of the printed hydrogels showed significant changes above the LCST: storage- and loss moduli both increased and loss tangent and compressive modulus decreased above this temperature (P ≤ 0.01). The highly cytocompatible hydrogel microwell arrays yielded both single- and multilayer spheroids with human dermal fibroblasts (HDFs) and HeLa cells successfully. Evaluation of the release of encapsulated model macro- (bovine serum albumin, BSA) and small molecule (rhodamine B) drugs in a buffer solution showed an interestingly inverted thermoresponsive release profile with >80% release at room temperature and about 50-60% release above the gels' LCST. All told, the optimized ink holds great promise for multiple biomedical applications including precise and high-resolution fabrication of complex tissue structures, development of smart drug delivery systems and 3D cell culture.

3D Bioprinted Implants for Cartilage Repair in Intervertebral Discs and Knee Menisci.

Cartilage defects pose a significant clinical challenge as they can lead to joint pain, swelling and stiffness, which reduces mobility and function thereby significantly affecting the quality of life of patients. More than 250,000 cartilage repair surgeries are performed in the United States every year. The current gold standard is the treatment of focal cartilage defects and bone damage with nonflexible metal or plastic prosthetics. However, these prosthetics are often made from hard and stiff materials that limits mobility and flexibility, and results in leaching of metal particles into the body, degeneration of adjacent soft bone tissues and possible failure of the implant with time. As a result, the patients may require revision surgeries to replace the worn implants or adjacent vertebrae. More recently, autograft - and allograft-based repair strategies have been studied, however these too are limited by donor site morbidity and the limited availability of tissues for surgery. There has been increasing interest in the past two decades in the area of cartilage tissue engineering where methods like 3D bioprinting may be implemented to generate functional constructs using a combination of cells, growth factors (GF) and biocompatible materials. 3D bioprinting allows for the modulation of mechanical properties of the developed constructs to maintain the required flexibility following implantation while also providing the stiffness needed to support body weight. In this review, we will provide a comprehensive overview of current advances in 3D bioprinting for cartilage tissue engineering for knee menisci and intervertebral disc repair. We will also discuss promising medical-grade materials and techniques that can be used for printing, and the future outlook of this emerging field.

Fabrication of PNIPAm-based thermoresponsive hydrogel microwell arrays for tumor spheroid formation.

Complex three-dimensional (3D) cell cultures are being increasingly implemented in biomedical research as they provide important insights into complex cancer biology, and cell-cell and cell-matrix interactions in the tumor microenvironment. However, most methods used today for 3D cell culture are limited by high cost, the need for specialized skills, low throughput and the use of unnatural culture environments. We report the development of a unique biomimetic hydrogel microwell array platform for the generation and stress-free isolation of cancer spheroids. The poly N-isopropylacrylamide-based hydrogel microwell array (PHMA) has thermoresponsive properties allowing for the attachment and growth of cell aggregates/ spheroids at 37 °C, and their easy isolation at room temperature (RT). The reversible phase transition of the microwell arrays at 35 °C was confirmed visually and by differential scanning calorimetry. Swelling/ shrinking studies and EVOS imaging established that the microwell arrays are hydrophilic and swollen at temperatures <35 °C, while they shrink and are hydrophobic at temperatures >35 °C. Spheroid development within the PHMA was optimized for seeding density, incubation time and cell viability. Spheroids of A549, HeLa and MG-63 cancer cell lines, and human lung fibroblast (HLF) cell line generated within the PHMAs had relatively spherical morphology with hypoxic cores. Finally, using MG-63 cell spheroids as representative models, a proof-of-concept drug response study using doxorubicin hydrochloride was conducted. Overall, we demonstrate that the PHMAs are an innovative alternative to currently used 3D cell culture techniques, for the high-throughput generation of cell spheroids for disease modeling and drug screening applications.