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Biomedical subjects

Robert Langer

Publications and source records attributed to Robert Langer.

At least 19 recordsLinked to original sources

The Use of Deep Learning in RNA Therapeutic Development.

Ribonucleic acid (RNA)-based therapeutics have emerged as promising methods of disease treatment due to their ability to target the human genome and influence protein production, their versatility, and their relative lack of toxicity compared to other gene therapies. However, the RNA therapeutic design space is extremely large, encompassing multiple variables, including codon identities, secondary structure, and design of specific regions. RNA therapeutic optimization is difficult due to the impracticality of exploring such a vast design space experimentally. To address this limitation, deep learning methods have been employed to optimize RNA therapeutic development. In this review, we examine the application of deep learning models across three key aspects of RNA therapeutic development (RNA structure prediction, CRISPR activity, and RNA delivery), highlighting major contributions in these fields and analyzing how deep learning model architectures could affect model performance. We then discuss challenges associated with using deep learning for RNA therapeutics, such as computational and data limitations. Finally, we offer perspectives on areas for future exploration, such as emerging model architectures and methods of integration with more advanced high-throughput screening techniques. Ultimately, this review provides an overview of how deep learning is used in RNA therapeutic development and how it can evolve in the future.

Deep Learning↗

Microfluidic patterning for fabrication of contractile cardiac organoids.

The development of in vitro methods of engineering three-dimensional cardiac tissues can be useful for tissue replacement, diagnostics and drug discovery. Here, we introduce the use of patterned hyaluronic acid (HA) substrates generated using microfluidic patterning as a method of fabricating 3D cardiac organoids. HA micropatterns served as inductive templates for organoid assembly. Upon seeding, cardiomyocytes elongated and aligned along the pattern direction attaching preferentially to the glass substrate and the interface between HA patterns and glass substrate. After 3 days in culture, the linearly aligned myocytes detached from the surface and formed contractile cardiac organoids. The procedure can be utilized to simply, rapidly and inexpensively create in vitro cardiac tissue models.

Animals↗

Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment.

Micropatterning of hydrogels is potentially useful for a variety of applications, including tissue engineering, fundamental biological studies, diagnostics, and high-throughput screening. Although synthetic polymers have been developed for these applications, natural polymers such as polysaccharides may have advantages for biological samples and cell-based devices because they are natural components of the in vivo microenvironment. In this study, we synthesized and used hyaluronic acid (HA) modified with photoreactive methacrylates to fabricate microstructures as functional components of microfabricated devices. To demonstrate the universality of this approach, two types of microstructures were formed. In the first approach, HA microstructures were fabricated and used as docking templates to enable the subsequent formation of cell microarrays within low shear stress regions of the patterns. Cells within these microwells remained viable, could generate spheroids, and could be retrieved using mechanical disruption. In the second approach, cells were encapsulated directly within the HA hydrogels. Arrays of viable embryonic stem (ES) cells or fibroblasts were encapsulated within HA hydrogels and could later be recovered using enzymatic digestion of the microstructures. These results demonstrate that it is possible to incorporate photocrosslinkable HA, a natural, versatile, degradable, and biocompatible biopolymer, into micro-electromechanical systems.

Animals↗

Why inhaling salt water changes what we exhale.

We find that inhaling salt water diminishes subsequently exhaled biomaterial in man and animals due to reversible stabilization of the airway lining fluid (ALF)/air interface as a novel potential means for control of the spread of airborne infectious disease. The mechanism of this phenomenon relates to charge shielding of mucin or mucin-like macromolecules that consequently undergo gelation; this gelation alters the physical properties of the ALF surface and reduces its breakup. Cations in the nebulized solution and apparent surface viscoelasticity of the ALF (more than any other ALF intrinsic physical property) appear to be responsible for the reduced tendency of the ALF to disintegrate into very small droplets. We confirm these effects in vivo and show their reversibility through nebulization of saline solutions to anesthetized bull calves.

Administration, Inhalation↗

Nanomedicine: developing smarter therapeutic and diagnostic modalities.

The early impact of nanotechnology on medicine is beginning to get realized, with novel nanoscale therapeutic and diagnostic modalities under development or in clinical practice today. In this commentary the field of "nanomedicine" is briefly reviewed form the perspective of where we were; where we are today; and where we are likely to go tomorrow.

Diagnostic Techniques and Procedures↗

Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery.

Nanoparticle (NP) size has been shown to significantly affect the biodistribution of targeted and non-targeted NPs in an organ specific manner. Herein we have developed NPs from carboxy-terminated poly(d,L-lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG-COOH) polymer and studied the effects of altering the following formulation parameters on the size of NPs: (1) polymer concentration, (2) drug loading, (3) water miscibility of solvent, and (4) the ratio of water to solvent. We found that NP mean volumetric size correlates linearly with polymer concentration for NPs between 70 and 250 nm in diameter (linear coefficient=0.99 for NPs formulated with solvents studied). NPs with desirable size, drug loading, and polydispersity were conjugated to the A10 RNA aptamer (Apt) that binds to the prostate specific membrane antigen (PSMA), and NP and NP-Apt biodistribution was evaluated in a LNCaP (PSMA+) xenograft mouse model of prostate cancer. The surface functionalization of NPs with the A10 PSMA Apt significantly enhanced delivery of NPs to tumors vs. equivalent NPs lacking the A10 PSMA Apt (a 3.77-fold increase at 24h; NP-Apt 0.83%+/-0.21% vs. NP 0.22%+/-0.07% of injected dose per gram of tissue; mean+/-SD, n=4, p=0.002). The ability to control NP size together with targeted delivery may result in favorable biodistribution and development of clinically relevant targeted therapies.

Animals↗

Synthesis of poly(beta-amino ester)s with thiol-reactive side chains for DNA delivery.

The safe and efficient delivery of DNA remains the major barrier to the clinical application of non-viral gene therapy. Here, we present novel, biodegradable polymers for gene delivery that are capable of simple graft modification and demonstrate the ability to respond to intracellular conditions. We synthesized poly(beta-amino ester)s using a new amine monomer, 2-(pyridyldithio)-ethylamine (PDA). These cationic, degradable polymers contain pyridyldithio functionalities in the side chains that react with high specificity toward thiol ligands. This reactivity is demonstrated using both mercaptoethylamine (MEA) and the thiol peptide RGDC, a ligand that binds with high affinity to certain integrin receptors. These two polymer derivatives displayed strong DNA binding as determined using electrophoresis and dye exclusion assays. In addition, the MEA-based polymer and plasmid DNA were shown to self-assemble into cationic complexes with effective diameters as low as 100 nm. Furthermore, this DNA binding ability was substantially reduced in response to intracellular glutathione concentrations, which may aid in DNA unpackaging inside the cell. These complexes also displayed low cellular toxicity and were able to mediate transfection at levels comparable to PEI in human hepatocellular carcinoma cells. These results suggest that PDA-based poly(beta-amino ester)s may serve as a modular platform for polymer-mediated gene delivery.

Biocompatible Materials↗

Peritoneal application of chitosan and UV-cross-linkable chitosan.

The suitability of chitosan and UV-cross-linkable chitosan for intraperitoneal use, for example as a barrier device for preventing peritoneal adhesions or for drug delivery, was examined. In vitro experiments using two major cell types present in the peritoneal cavity (mesothelial cells and peritoneal macrophages) revealed neither attractive interactions between cross-linked chitosan gels and the cells nor a proliferative effect. However, the same UV-cross-linked chitosan applied in the peritoneal cavity of rabbits caused a granulomatous reaction with adhesion formation within two weeks in all animals, which persisted up to 4 weeks after exposure. Unmodified chitosan also caused adhesions, while UV irradiation did not. UV-cross-linkable chitosan induced significant elevations in MIP-2 and TNF-alpha from peritoneal macrophages, suggesting that soluble mediators could play a role in inducing adhesion formation. These results reinforce the view that the predictive value of in vitro cytotoxicity assays in matters of biocompatibility may not be sufficient, and suggest that other assays such as cytokine levels may be of value in predicting outcomes in situations involving multiple cell types (i.e. in vivo).

Animals↗

Co-culture of human embryonic stem cells with murine embryonic fibroblasts on microwell-patterned substrates.

Human embryonic stem (hES) cells are generally cultured as cell clusters on top of a feeder layer formed by mitotically inactivated murine embryonic fibroblasts (MEFs) to maintain their undifferentiated state. This co-culture system, which is typically used to expand the population of undifferentiated hES cells, presents several challenges since it is difficult to control cell cluster size. Large cell clusters tend to differentiate at the borders, and clusters with different sizes may lead to heterogeneous differentiation patterns within embryoid bodies. In this work, we develop a new approach to culture hES cells with controlled cluster size and number through merging microfabrication, and biomaterials technologies. Polymeric microwells were fabricated and used to control the size and uniformity of hES cell clusters in co-culture with MEFs. The results show that it is possible to culture hES cells homogeneously while keeping their undifferentiated state as confirmed by the expression of stem cell markers octamer binding protein 4 (Oct-4) and alkaline phosphatase (ALP). In addition, these clusters can be recovered from the microwells to generate nearly homogeneous cell aggregates for differentiation experiments.

Animals↗

Heparin localization and fine structure regulate Burkitt's lymphoma growth.

Burkitt's lymphoma (BL) is a B-cell malignancy associated with the Epstein-Barr virus (EBV). Mounting evidence has implicated heparan sulfate proteoglycans and heparan sulfate-like glycosaminoglycans (HSGAGs) in the initiation, severity, and progression of the malignancy. The importance of HSGAGs in regulating BL cell growth was therefore examined. Extracellular exogenous heparin inhibited cell growth >30%, while heparin internalized with poly(beta-amino ester)s promoted proliferation up to 58%. The growth-modulating effects of heparin and internalized heparin were dependent on cell surface HSGAGs, PI3K, and Erk/Mek. Treatment of cells with protamine sulfate or with heparinases potently inhibited proliferation, with the greatest effects induced by heparinase I. Cell surface HSGAGs therefore play an important role in regulating BL proliferation and may offer a potential target for therapeutic intervention.

Burkitt Lymphoma↗

Poly(ethylene oxide)-modified poly(beta-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs: part 3. Therapeutic efficacy and safety studies in ovarian cancer xenograft model.

PURPOSE: The objective of this study was to evaluate the anti-tumor efficacy and lack of systemic toxicity of paclitaxel when administered in pH-sensitive poly(ethylene oxide) (PEO)-modified poly(beta-amino ester) (PbAE) nanoparticles in mice bearing human ovarian adenocarcinoma (SKOV-3) xenograft. METHODS: Paclitaxel-encapsulated PEO-modified PbAE (PEO-PbAE) nanoparticles were prepared by the solvent displacement method. PEO-modified poly(epsilon-caprolactone) (PCL) (PEO-PCL) nanoparticles were used as a non pH-responsive control formulation. Efficacy studies were conducted in SKOV-3 tumor-bearing athymic (Nu/Nu) mice at an equivalent paclitaxel dose of 20 mg/kg with the control and nanoparticle formulations. Safety of the drug when administered in the control and nanoparticle formulation was determined from blood cell counts and changes in body weight of the animals. RESULTS: The formulated paclitaxel-containing PEO-PbAE and PEO-PCL nanoparticles had a particle size in the range of 100-200 nm and a surface charge of + 39.0 and - 30.8 mV, respectively. After intravenous administration of paclitaxel in these formulations, the tumor growth was inhibited significantly. Both of the formulated nanoparticles tested have shown improved therapeutic efficacy as compared to the paclitaxel aqueous solution. Additionally, significantly lower toxicity profile of paclitaxel was observed with PEO-modified nanoparticles as compared to the aqueous solution formulation CONCLUSION: PEO-modified PbAE nanoparticles are a unique pH-sensitive drug delivery system that elicits enhanced efficacy and safety profile in solid tumor therapy.

Animals↗

The association between aspirin use and the incidence of colorectal cancer in women.

The purpose of this study was to test the hypothesis that aspirin use is associated with a decreased risk of incident colorectal cancer. From the Women's Health Initiative, 91,574 participants between the ages of 50 and 79 years at baseline in 1993-1998 provided details on aspirin use via interview using a standardized questionnaire and were subsequently followed annually for incident colorectal cancer during a period of over 6 years. For those persons who reported aspirin use, the type of compound, dose, and duration of use were recorded. Medical histories suggestive of colorectal cancers at the annual update were verified by medical record and pathology report review by trained local physician adjudicators. There were 631 confirmed cases of invasive cancer of the colon or rectum. There was no significant association between any aspirin use and risk for incident colorectal cancer (hazard ratio = 0.96, 95% confidence interval: 0.8, 1.2). Moreover, with no aspirin use as the referent category, there were no significant associations for duration of aspirin intake by category (< 1, 1- < 2, 2- < 3, 3- < 4, 4- < 5, and > or = 5 years) or for daily dosage by category (< 165, 165- < 300, 300- < 495, or > or = 495 mg).

Aged↗

Micromolding of shape-controlled, harvestable cell-laden hydrogels.

Encapsulation of mammalian cells within hydrogels has great utility for a variety of applications ranging from tissue engineering to cell-based assays. In this work, we present a technique to encapsulate live cells in three-dimensional (3D) microscale hydrogels (microgels) of controlled shapes and sizes in the form of harvestable free standing units. Cells were suspended in methacrylated hyaluronic acid (MeHA) or poly(ethylene glycol) diacrylate (PEGDA) hydrogel precursor solution containing photoinitiator, micromolded using a hydrophilic poly(dimethylsiloxane) (PDMS) stamp, and crosslinked using ultraviolet (UV) radiation. By controlling the features on the PDMS stamp, the size and shape of the molded hydrogels were controlled. Cells within microgels were well distributed and remained viable. These shape-specific microgels could be easily retrieved, cultured and potentially assembled to generate structures with controlled spatial distribution of multiple cell types. Further development of this technique may lead to applications in 3D co-cultures for tissue/organ regeneration and cell-based assays in which it is important to mimic the architectural intricacies of physiological cell-cell interactions.

Animals↗

Micromolding of photocrosslinkable chitosan hydrogel for spheroid microarray and co-cultures.

Bioengineering approaches, such as co-cultures of multiple cell types, that aim to mimic the physiological microenvironment may be beneficial for optimizing cell function and for engineering tissues in vitro. This study describes a novel method for preparing a spheroid microarray on microfabricated hydrogels, alone or in co-cultures. Photocrosslinkable chitosan was synthesized and utilized for fabricating hydrogel microstructures through a micromolding process. The chitosan surface was initially cell repellent but became increasingly cell adhesive over time. By using this unique property of chitosan hydrogels, it was possible to generate patterned co-cultures of spheroids and support cells. In this scheme, cells were initially microarrayed within low shear stress regions of microwells. Human hepatoblastoma cells, Hep G2, seeded in these wells formed spheroids with controlled sizes and shapes and stably secreted albumin during the culture period. The change of cell adhesive properties in the chitosan surface facilitated the adhesion and growth of a second cell type, NIH-3T3 fibroblast, and therefore enabled co-cultures of hepatocyte spheroids and fibroblast monolayers. This co-culture system could be a useful platform for studying heterotypic cell-cell interactions, for drug screening, and for developing implantable bioartificial organs.

Animals↗

Osteogenesis by human mesenchymal stem cells cultured on silk biomaterials: comparison of adenovirus mediated gene transfer and protein delivery of BMP-2.

Bone tissue engineering, gene therapy based on human mesenchymal stem cells (MSCs) and silk fibroin biomaterials were combined to study the impact of viral transfection on MSC osteogenic performance in vitro. MSCs were transduced with adenovirus containing a human BMP-2 (Ad-BMP-2) gene at clinically reasonable viral concentrations and cultured for 4 weeks. Controls with nontransfected MSCs, but exposed to exogenous BMP-2 concentrations on an analogous time profile as that secreted by the Ad-BMP-2 group, were compared. Both the Ad-BMP-2 MSC group and the exogenous protein BMP-2 group strongly expressed osteopontin and bone sialoprotein. Cells secreted a matrix that underwent mineralization on the silk fibroin scaffolds, forming clusters of osseous material, as determined by micro-computed tomography. The expression of osteogenic marker proteins and alkaline phosphatase was significantly higher in the Ad-BMP-2 MSC group than in the exogenous protein BMP-2 group, and no significant differences in mineralization were observed in two of the three MSC sources tested. The results demonstrate that transfection resulted in higher levels of expression of osteogenic marker genes, no change in proliferation rate and did not impact the capacity of the cells to calcify tissues on these protein scaffolds. These findings suggest additional options to control differentiation where exogenous additions of growth factors or morphogens can be replaced with transfected MSCs.

Adenoviridae↗

In situ cross-linkable hyaluronic acid hydrogels prevent post-operative abdominal adhesions in a rabbit model.

We studied the efficacy of an in situ cross-linked hyaluronic acid hydrogel (HAX) in preventing post-surgical peritoneal adhesions, using a rabbit sidewall defect-cecum abrasion model. Two cross-linkable precursors were prepared by modifying hyaluronic acid with adipic dihydrazide and aldehyde, respectively. The hydrogel precursors cross-linked to form a flexible hydrogel upon mixing. The hydrogel was biodegradable and provided a durable physical barrier, which was highly effective in reducing the formation of post-operative adhesions. Ten out of 12 animals in the untreated control group developed fibrous adhesions requiring sharp dissection, while only 2 out of 8 animals treated with HAX gels showed such adhesions, and those occurred in locations that were not covered by the hydrogel. We also studied means by which gel degradation time can be modulated by varying the precursor concentration and molecular weight.

Abdomen↗