On-Demand Drug Release from Click-Refillable Drug Depots.

Stimuli-responsive, on-demand release of drugs from drug-eluting depots could transform the treatment of many local diseases, providing intricate control over local dosing. However, conventional on-demand drug release approaches rely on locally implanted drug depots, which become spent over time and cannot be refilled or reused without invasive procedures. New strategies to noninvasively refill drug-eluting depots followed by on-demand release could transform clinical therapy. Here we report an on-demand drug delivery paradigm that combines bioorthogonal click chemistry to locally enrich protodrugs at a prelabeled site and light-triggered drug release at the target tissue. This approach begins with introduction of the targetable depot through local injection of chemically reactive azide groups that anchor to the extracellular matrix. The anchored azide groups then capture blood-circulating protodrugs through bioorthogonal click chemistry. After local capture and retention, active drugs can be released through external light irradiation. In this report, a photoresponsive protodrug was constructed consisting of the chemotherapeutic doxorubicin (Dox), conjugated to dibenzocyclooctyne (DBCO) through a photocleavable ortho-nitrobenzyl linker. The protodrug exhibited excellent on-demand light-triggered Dox release properties and light-mediated in vitro cytotoxicity in U87 glioblastoma cell lines. Furthermore, in a live animal setting, azide depots formed in mice through intradermal injection of activated azide-NHS esters. After i.v. administration, the protodrug was captured by the azide depots with intricate local specificity, which could be increased with multiple refills. Finally, doxorubicin could be released from the depot upon light irradiation. Multiple rounds of depot refilling and light-mediated release of active drug were accomplished, indicating that this system has the potential for multiple rounds of treatment. Taken together, these in vitro and in vivo proof of concept studies establish a novel method for in vivo targeting and on-demand delivery of cytotoxic drugs at target tissues.

Implantable CAR T cell factories enhance solid tumor treatment.

Chimeric Antigen Receptor (CAR) T cell therapy has produced revolutionary success in hematological cancers such as leukemia and lymphoma. Nonetheless, its translation to solid tumors faces challenges due to manufacturing complexities, short-lived in vivo persistence, and transient therapeutic impact. We introduce 'Drydux' - an innovative macroporous biomaterial scaffold designed for rapid, efficient in-situ generation of tumor-specific CAR T cells. Drydux expedites CAR T cell preparation with a mere three-day turnaround from patient blood collection, presenting a cost-effective, streamlined alternative to conventional methodologies. Notably, Drydux-enabled CAR T cells provide prolonged in vivo release, functionality, and enhanced persistence exceeding 150 days, with cells transitioning to memory phenotypes. Unlike conventional CAR T cell therapy, which offered only temporary tumor control, equivalent Drydux cell doses induced lasting tumor remission in various animal tumor models, including systemic lymphoma, peritoneal ovarian cancer, metastatic lung cancer, and orthotopic pancreatic cancer. Drydux's approach holds promise in revolutionizing solid tumor CAR T cell therapy by delivering durable, rapid, and cost-effective treatments and broadening patient accessibility to this groundbreaking therapy.

A biomaterial platform for T cell-specific gene delivery.

Efficient T cell engineering is central to the success of CAR T cell therapy but involves multiple time-consuming manipulations, including T cell isolation, activation, and transduction. These steps add complexity and delay CAR T cell manufacturing, which takes a mean time of 4 weeks. To streamline T cell engineering, we strategically combine two critical engineering solutions - T cell-specific lentiviral vectors and macroporous scaffolds - that enable T cell activation and transduction in a simple, single step. The T cell-specific lentiviral vectors (referred to as STAT virus) target T cells through the display of an anti-CD3 antibody and the CD80 extracellular domain on their surface and provide robust T cell activation. Biocompatible macroporous scaffolds (referred to as Drydux) mediate robust transduction by providing effective interaction between naïve T cells and viral vectors. We show that when unstimulated peripheral blood mononuclear cells (PBMCs) are seeded together with STAT lentivirus on Drydux scaffolds, T cells are activated, selectively transduced, and reprogrammed in a single step. Further, we show that the Drydux platform seeded with PBMCs and STAT lentivirus generates tumor-specific functional CAR T cells. This potent combination of engineered lentivirus and biomaterial scaffold holds promise for an effective, simple, and safe avenue for in vitro and in vivo T cell engineering. STATEMENT OF SIGNIFICANCE: Manufacturing T cell therapies involves lengthy and labor-intensive steps, including T cell selection, activation, and transduction. These steps add complexity to current CAR T cell manufacturing protocols and limit widespread patient access to this revolutionary therapy. In this work, we demonstrate the combination of engineered virus and biomaterial platform that, together, enables selective T cell activation and transduction in a single step, eliminating multistep T cell engineering protocols and significantly simplifying the manufacturing process.

Absorption rate governs cell transduction in dry macroporous scaffolds.

Developing the next generation of cellular therapies will depend on fast, versatile, and efficient cellular reprogramming. Novel biomaterials will play a central role in this process by providing scaffolding and bioactive signals that shape cell fate and function. Previously, our lab reported that dry macroporous alginate scaffolds mediate retroviral transduction of primary T cells with efficiencies that rival the gold-standard clinical spinoculation procedures, which involve centrifugation on Retronectin-coated plates. This scaffold transduction required the scaffolds to be both macroporous and dry. Transduction by dry, macroporous scaffolds, termed "Drydux transduction," provides a fast and inexpensive method for transducing cells for cellular therapy, including for the production of CAR T cells. In this study, we investigate the mechanism of action by which Drydux transduction works through exploring the impact of pore size, stiffness, viral concentration, and absorption speed on transduction efficiency. We report that Drydux scaffolds with macropores ranging from 50-230 µm and with Young's moduli ranging from 25-620 kPa all effectively transduce primary T cells, suggesting that these parameters are not central to the mechanism of action, but also demonstrating that Drydux scaffolds can be tuned without losing functionality. Increasing viral concentrations led to significantly higher transduction efficiencies, demonstrating that increased cell-virus interaction is necessary for optimal transduction. Finally, we discovered that the rate with which the cell-virus solution is absorbed into the scaffold is closely correlated to viral transduction efficiency, with faster absorption producing significantly higher transduction. A computational model of liquid flow through porous media validates this finding by showing that increased fluid flow substantially increases collisions between virus particles and cells in a porous scaffold. Taken together, we conclude that the rate of liquid flow through the scaffolds, rather than pore size or stiffness, serves as a central regulator for efficient Drydux transduction.

Fabrication and Use of Dry Macroporous Alginate Scaffolds for Viral Transduction of T Cells.

Genetic engineering of T cells for CAR-T cell therapy has come to the forefront of cancer treatment over the last few years. CAR-T cells are produced by viral gene transfer into T cells. The current gold standard of viral gene transfer involves spinoculation of retronectin-coated plates, which is expensive and time-consuming. There is a significant need for efficient and cost-effective methods to generate CAR-T cells. Described here is a method for fabricating inexpensive, dry macroporous alginate scaffolds, known as Drydux scaffolds, that efficiently promote viral transduction of activated T cells. The scaffolds are designed to be used in place of gold standard spinoculation of retronectin-coated plates seeded with virus and simplify the process for transducing cells. Alginate is cross-linked with calcium-D-gluconate and frozen overnight to create the scaffolds. The frozen scaffolds are freeze-dried in a lyophilizer for 72 h to complete the formation of the dry macroporous scaffolds. The scaffolds mediate viral gene transfer when virus and activated T cells are seeded together on top of the scaffold to produce genetically modified cells. The scaffolds produce >85% primary T cell transduction, which is comparable to the transduction efficiency of spinoculation on retronectin-coated plates. These results demonstrate that dry macroporous alginate scaffolds serve as a cheaper and more convenient alternative to the conventional transduction method.

Tissue-reactive drugs enable materials-free local depots.

Local, sustained drug delivery of potent therapeutics holds promise for the treatment of a myriad of localized diseases while eliminating systemic side effects. However, introduction of drug delivery depots such as viscous hydrogels or polymer-based implants is highly limited in stiff tissues such as desmoplastic tumors. Here, we present a method to create materials-free intratumoral drug depots through Tissue-Reactive Anchoring Pharmaceuticals (TRAPs). TRAPs diffuse into tissue and attach locally for sustained drug release. In TRAPs, potent drugs are modified with ECM-reactive groups and then locally injected to quickly react with accessible amines within the ECM, creating local drug depots. We demonstrate that locally injected TRAPs create dispersed, stable intratumoral depots deep within mouse and human pancreatic tumor tissues. TRAPs depots based on ECM-reactive paclitaxel (TRAP paclitaxel) had better solubility than free paclitaxel and enabled sustained in vitro and in vivo drug release. TRAP paclitaxel induced higher tumoral apoptosis and sustained better antitumor efficacy than the free drug. By providing continuous drug access to tumor cells, this material-free approach to sustained drug delivery of potent therapeutics has the potential in a wide variety of diseases where current injectable depots fall short.