Design and evaluation of nanoscale materials with programmed responsivity towards epigenetic enzymes.

Self-assembled materials capable of modulating their assembly properties in response to specific enzymes play a pivotal role in advancing 'intelligent' encapsulation platforms for biotechnological applications. Here, we introduce a previously unreported class of synthetic nanomaterials that programmatically interact with histone deacetylase (HDAC) as the triggering stimulus for disassembly. These nanomaterials consist of co-polypeptides comprising poly(acetyl L-lysine) and poly(ethylene glycol) blocks. Under neutral pH conditions, they self-assemble into particles. The hydrodynamic diameters of particles were typically withing the range of 108-190 nm, depending on degree of acetylation of the hydrophobic block. However, their stability is compromised upon exposure to HDACs, depending on enzyme concentration and exposure time. Our investigation, utilizing HDAC8 as the model enzyme, revealed that the primary mechanism behind disassembly involves a decrease in amphiphilicity within the block copolymer due to the deacetylation of lysine residues within the particles' hydrophobic domains. To elucidate the response mechanism, we encapsulated a fluorescent dye within these nanoparticles. Upon incubation with HDAC, the nanoparticle structure collapsed, leading to controlled release of the dye over time. Notably, this release was not triggered by denatured HDAC8, other proteolytic enzymes like trypsin, or the co-presence of HDAC8 and its inhibitor. We also demonstrated the biocompatibility and cellular effects of these materials in the context of drug delivery in different types of anticancer cell lines, such as MIA PaCa-2, PANC-1, cancer like stem cells (CSCs), and non-cancerous HPNE cells. We observed that the release of a model drug (such as a STAT3 pathway inhibitor, Napabucasin) can be loaded into these nanoparticles, with >90% of the dosage can be released over 3 h under the influence of HDAC8 enzyme in a controlled fashion. Further, we conducted a comprehensive computational study to unveil the possible interaction mechanism between enzymes and particles. By drawing parallels to the mechanism of naturally occurring histone proteins, this research represents a pioneering step toward developing functional materials capable of harnessing the activity of epigenetic enzymes such as HDACs.

Design and Evaluation of Nanoscale Materials with Programmed Responsivity towards Epigenetic Enzymes.

Self-assembled materials capable of modulating their assembly properties in response to specific enzymes play a pivotal role in advancing 'intelligent' encapsulation platforms for biotechnological applications. Here, we introduce a previously unreported class of synthetic nanomaterials that programmatically interact with histone deacetylase (HDAC) as the triggering stimulus for disassembly. These nanomaterials consist of co-polypeptides comprising poly (acetyl L-lysine) and poly(ethylene glycol) blocks. Under neutral pH conditions, they self-assemble into particles. However, their stability is compromised upon exposure to HDACs, depending on enzyme concentration and exposure time. Our investigation, utilizing HDAC8 as the model enzyme, revealed that the primary mechanism behind disassembly involves a decrease in amphiphilicity within the block copolymer due to the deacetylation of lysine residues within the particles' hydrophobic domains. To elucidate the response mechanism, we encapsulated a fluorescent dye within these nanoparticles. Upon incubation with HDAC, the nanoparticle structure collapsed, leading to controlled release of the dye over time. Notably, this release was not triggered by denatured HDAC8, other proteolytic enzymes like trypsin, or the co-presence of HDAC8 and its inhibitor. We further demonstrated the biocompatibility and cellular effects of these materials and conducted a comprehensive computational study to unveil the possible interaction mechanism between enzymes and particles. By drawing parallels to the mechanism of naturally occurring histone proteins, this research represents a pioneering step toward developing functional materials capable of harnessing the activity of epigenetic enzymes such as HDACs.

Acquired αSMA Expression in Pericytes Coincides with Aberrant Vascular Structure and Function in Pancreatic Ductal Adenocarcinoma.

The subpopulations of tumor pericytes undergo pathological phenotype switching, affecting their normal function in upholding structural stability and cross-communication with other cells. In the case of pancreatic ductal adenocarcinoma (PDAC), a significant portion of blood vessels are covered by an α-smooth muscle actin (αSMA)-expressing pericyte, which is normally absent from capillary pericytes. The DesminlowαSMAhigh phenotype was significantly correlated with intratumoral hypoxia and vascular leakiness. Using an in vitro co-culture system, we demonstrated that cancer cell-derived exosomes could induce ectopic αSMA expression in pericytes. Exosome-treated αSMA+ pericytes presented altered pericyte markers and an acquired immune-modulatory feature. αSMA+ pericytes were also linked to morphological and biomechanical changes in the pericyte. The PDAC exosome was sufficient to induce αSMA expression by normal pericytes of the healthy pancreas in vivo, and the vessels with αSMA+ pericytes were leaky. This study demonstrated that tumor pericyte heterogeneity could be dictated by cancer cells, and a subpopulation of these pericytes confers a pathological feature.

Modified Bovine Milk Exosomes for Doxorubicin Delivery to Triple-Negative Breast Cancer Cells.

Biological nanoparticles, such as exosomes, offer an approach to drug delivery because of their innate ability to transport biomolecules. Exosomes are derived from cells and an integral component of cellular communication. However, the cellular cargo of human exosomes could negatively impact their use as a safe drug carrier. Additionally, exosomes have the intrinsic yet enigmatic, targeting characteristics of complex cellular communication. Hence, harnessing the natural transport abilities of exosomes for drug delivery requires predictably targeting these biological nanoparticles. This manuscript describes the use of two chemical modifications, incorporating a neuropilin receptor agonist peptide (iRGD) and a hypoxia-responsive lipid for targeting and release of an encapsulated drug from bovine milk exosomes to triple-negative breast cancer cells. Triple-negative breast cancer is a very aggressive and deadly form of malignancy with limited treatment options. Incorporation of both the iRGD peptide and hypoxia-responsive lipid into the lipid bilayer of bovine milk exosomes and encapsulation of the anticancer drug, doxorubicin, created the peptide targeted, hypoxia-responsive bovine milk exosomes, iDHRX. Initial studies confirmed the presence of iRGD peptide and the exosomes' ability to target the αvß3 integrin, overexpressed on triple-negative breast cancer cells' surface. These modified exosomes were stable under normoxic conditions but fragmented in the reducing microenvironment created by 10 mM glutathione. In vitro cellular internalization studies in monolayer and three-dimensional (3D) spheroids of triple-negative breast cancer cells confirmed the cell-killing ability of iDHRX. Cell viability of 50% was reached at 10 µM iDHRX in the 3D spheroid models using four different triple-negative breast cancer cell lines. Overall, the tumor penetrating, hypoxia-responsive exosomes encapsulating doxorubicin would be effective in reducing triple-negative breast cancer cells' survival.

Single-Molecule Force Probing of RGD-Binding Integrins on Pancreatic Cancer Cells.

Integrin-targeting arginine-glycine-aspartic acid (RGD)-based nanocarriers have been widely used for tumor imaging, monitoring of tumor development, and delivery of anticancer drugs. However, the thermodynamics of an RGD-integrin formation and dissociation associated with binding dynamics, affinity, and stability remains unclear. Here, we probed the binding strength of the binary complex to live pancreatic cancer cells using single-molecule binding force spectroscopy methods, in which RGD peptides were functionalized on a force probe tip through poly(ethylene glycol) (PEG)-based bifunctional linker molecules. While the density of integrin αV receptors on the cell surface varies more than twofold from cell line to cell line, the individual RGD-integrin complexes exhibited a cell type-independent, monovalent bond strength. The load-dependent bond strength of multivalent RGD-integrin interactions scaled sublinearly with increasing bond number, consistent with the noncooperative, parallel bond model. Furthermore, the multivalent bonds ruptured sequentially either by one or in multiples, and the force strength was comparable to the synchronous rupture force. Comparison of energy landscapes of the bond number revealed a substantial decrease of kinetic off-rates for multivalent bonds, along with the increased width of the potential well and the increased potential barrier height between bound and unbound states, enhancing the stability of the multivalent bonds between them.

Dynamic cellular biomechanics in responses to chemotherapeutic drug in hypoxia probed by atomic force spectroscopy.

The changes in cellular structure play an important role in cancer cell development, progression, and metastasis. By exploiting single-cell, force spectroscopy methods, we probed biophysical and biomechanical kinetics (stiffness, morphology, roughness, adhesion) of brain, breast, prostate, and pancreatic cancer cells with standard chemotherapeutic drugs in normoxia and hypoxia over 12-24 hours. After exposure to the drugs, we found that brain, breast, and pancreatic cancer cells became approximately 55-75% less stiff, while prostate cancer cells became more stiff, due to either drug-induced disruption or reinforcement of cytoskeletal structure. However, the rate of the stiffness change decreased up to 2-folds in hypoxia, suggesting a correlation between cellular stiffness and drug resistance of cancer cells in hypoxic tumor microenvironment. Also, we observed significant changes in the cell body height, surface roughness, and cytoadhesion of cancer cells after exposure to drugs, which followed the trend of stiffness. Our results show that a degree of chemotherapeutic drug effects on biomechanical and biophysical properties of cancer cells is distinguishable in normoxia and hypoxia, which are correlated with alteration of cytoskeletal structure and integrity during drug-induced apoptotic process.

Size-Transformable, Multifunctional Nanoparticles from Hyperbranched Polymers for Environment-Specific Therapeutic Delivery.

Hyperbranched polymer-derived drug nanocarriers have been synthesized that can change sizes selectively in response to pH. These constructs were composed of tertiary amine-conjugated polycarbonate blocks "grafted from" a hyperbranched polyester polyol core. At neutral pH, unprotonated polycarbonate arms stabilized the copolymer aggregates in the form of nanoparticles with hydrodynamic diameters ranging from 150 to 190 nm. Upon lowering of pH, these larger aggregates disassembled into smaller nanoparticles with diameters of 3-5 nm as directed by protonation of tertiary amine side-chains. The pH-dependent reduction of particle sizes was evident by titrimetric, spectroscopic, dynamic light scattering, transmission electron, and atomic force microscopy-based experiments. We observed that these copolymeric nanoparticles could be loaded with dye and drug molecules either by noncovalent encapsulation or by covalent conjugation. A pH-induced disassembly of the aggregates initiated rapid release of the encapsulated payload, but not of the conjugated ones, thus establishing a controlled rate of therapeutic delivery from the nanostructures over an extended period. We envision that such systems can be used for drug delivery applications where nanoparticle sizes critically govern therapeutic efficiency in a vasculature-poor disease microenvironment such as desmoplasia in pancreatic cancer. Hence, we tested the cellular uptake, cytotoxicity, and chemotherapeutic potential of the size-modifiable nanoaggregates using gemcitabine as a model drug in pancreatic cancer setting. We observed that assembled nanoparticles were biocompatible to noncancerous cells, showed pH-dependent effects on cellular uptake as well as promoted accumulation within cancer cells cultured as 3D spheroids. We also found that when conjugated with gemcitabine, the resulting drug-loaded nanoparticles suppressed proliferation of cancer cells. Collectively, the studies suggested that these synthesized, pH-disassembling nanoscale platform will find applications as biomaterials for constructing a size-transformable drug nanocarriers where reduction of size takes effect near localized disease targets in response to microenvironmental triggers.