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.

pH-responsive targeted nanoparticles release ERK-inhibitor in the hypoxic zone and sensitize free gemcitabine in mutant K-Ras-addicted pancreatic cancer cells and mouse model.

Therapeutic options for managing Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest types of aggressive malignancies, are limited and disappointing. Therefore, despite suboptimal clinical effects, gemcitabine (GEM) remains the first-line chemotherapeutic drug in the clinic for PDAC treatment. The therapeutic limitations of GEM are primarily due to poor bioavailability and the development of chemoresistance resulting from the addiction of mutant-K-RAS/AKT/ERK signaling-mediated desmoplastic barriers with a hypoxic microenvironment. Several new therapeutic approaches, including nanoparticle-assisted drug delivery, are being investigated by us and others. This study used pH-responsive nanoparticles encapsulated ERK inhibitor (SCH772984) and surface functionalized with tumor-penetrating peptide, iRGD, to target PDAC tumors. We used a small molecule, SCH772984, to target ERK1 and ERK2 in PDAC and other cancer cells. This nanocarrier efficiently released ERKi in hypoxic and low-pH environments. We also found that the free-GEM, which is functionally weak when combined with nanoencapsulated ERKi, led to significant synergistic treatment outcomes in vitro and in vivo. In particular, the combination approaches significantly enhanced the GEM effect in PDAC growth inhibition and prolonged survival of the animals in a genetically engineered KPC (LSL-KrasG12D/+/LSL-Trp53R172H/+/Pdx-1-Cre) pancreatic cancer mouse model, which is not observed in a single therapy. Mechanistically, we anticipate that the GEM efficacy was increased as ERKi blocks desmoplasia by impairing the production of desmoplastic regulatory factors in PDAC cells and KPC mouse tumors. Therefore, 2nd generation ERKi (SCH 772984)-iRGD-pHNPs are vital for the cellular response to GEM and denote a promising therapeutic target in PDAC with mutant K-RAS.

Metal-Organic Framework Induced Stabilization of Proteins in Polymeric Nanoparticles.

Developing protein confinement platforms is an attractive research area that not only promotes protein delivery but also can result in artificial environment mimicking of the cellular one, impacting both the controlled release of proteins and the fundamental protein biophysics. Polymeric nanoparticles (PNPs) are attractive platforms to confine proteins due to their superior biocompatibility, low cytotoxicity, and controllable release under external stimuli. However, loading proteins into PNPs can be challenging due to the potential protein structural perturbation upon contacting the interior of PNPs. In this work, we developed a novel approach to encapsulate proteins in PNPs with the assistance of the zeolitic imidazolate framework (ZIF). Here, ZIF offers an additional protection layer to the target protein by forming the protein@ZIF composite via aqueous-phase cocrystallization. We demonstrated our platform using a model protein, lysozyme, and a widely studied PNP composed of poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA). A comprehensive study via standard loading and release tests as well as various spectroscopic techniques was carried out on lysozyme loaded onto PEG-PLGA with and without ZIF protection. As compared with the direct protein encapsulation, an additional layer with ZIF prior to loading offered enhanced loading capacity, reduced leaching, especially in the initial stage, led to slower release kinetics, and reduced secondary structural perturbation. Meanwhile, the function, cytotoxicity, and cellular uptake of proteins encapsulated within the ZIF-bound systems are decent. Our results demonstrated the use of ZIF in assisting in protein encapsulation in PNPs and established the basis for developing more sophisticated protein encapsulation platforms using a combination of materials of diverse molecular architectures and disciplines. As such, we anticipate that the protein-encapsulated ZIF systems will serve as future polymer protein confinement and delivery platforms for both fundamental biophysics and biochemistry research and biomedical applications where protein delivery is needed to support therapeutics and/or nutrients.

Benefits and Pitfalls of a Glycosylation Inhibitor Tunicamycin in the Therapeutic Implication of Cancers.

The aberrant glycosylation is a hallmark of cancer progression and chemoresistance. It is also an immune therapeutic target for various cancers. Tunicamycin (TM) is one of the potent nucleoside antibiotics and an inhibitor of aberrant glycosylation in various cancer cells, including breast cancer, gastric cancer, and pancreatic cancer, parallel with the inhibition of cancer cell growth and progression of tumors. Like chemotherapies such as doxorubicin (DOX), 5'fluorouracil, etoposide, and cisplatin, TM induces the unfolded protein response (UPR) by blocking aberrant glycosylation. Consequently, stress is induced in the endoplasmic reticulum (ER) that promotes apoptosis. TM can thus be considered a potent antitumor drug in various cancers and may promote chemosensitivity. However, its lack of cell-type-specific cytotoxicity impedes its anticancer efficacy. In this review, we focus on recent advances in our understanding of the benefits and pitfalls of TM therapies in various cancers, including breast, colon, and pancreatic cancers, and discuss the mechanisms identified by which TM functions. Finally, we discuss the potential use of nano-based drug delivery systems to overcome non-specific toxicity and enhance the therapeutic efficacy of TM as a targeted therapy.

Impact of Crystallinity on Enzyme Orientation and Dynamics upon Biomineralization in Metal-Organic Frameworks.

Aqueous-phase co-crystallization (also known as biomimetic mineralization or biomineralization) is a unique way to encapsulate large enzymes, enzyme clusters, and enzymes with large substrates in metal-organic frameworks (MOFs), broadening the application of MOFs as enzyme carriers. The crystallinity of resultant enzyme@MOF biocomposites, however, can be low, raising a concern about how MOF crystal packing quality affects enzyme performance upon encapsulation. The challenges to overcome this concern are (1) the limited database of enzyme performance upon biomineralization in different aqueous MOFs and (2) the difficulty in probing enzyme restriction and motion in the resultant MOF scaffolds, which are related to the local crystal packing quality/density, under the interference of the MOF backgrounds. We have discovered several new aqueous MOFs for enzyme biomineralization with varied crystallinity [Jordahl, D.; Armstrong, Z.; Li, Q.; Gao, R.; Liu, W.; Johnson, K.; Brown, W.; Scheiwiller, A.; Feng, L.; Ugrinov, A.; Mao, H.; Chen, B.; Quadir, M.; Pan, Y.; Li, H.; Yang, Z. Expanding the Library of Metal-Organic Frameworks (MOFs) for Enzyme Biomineralization. ACS Appl. Mater. Interfaces 2022, 14 (46), 51619-51629, DOI: 10.1021/acsami.2c12998]. Here, we address the second challenge by probing enzyme dynamics/restriction in these MOFs at the residue level via site-directed spin labeling (SDSL)-electron paramagnetic resonance (EPR) spectroscopy, a unique approach to determine protein backbone motions regardless of the background complexity. We encapsulated a model large-substrate enzyme, lysozyme, in eight newly discovered MOFs, which possess various degrees of crystallization, via aqueous-phase co-crystallization. Through the EPR study and simulations, we found rough connections between (a) enzyme mobility/dynamics and MOF crystal properties (packing quality and density) and (b) enzyme areas exposed above each MOF and their catalytic performance. This work suggests that protein SDSL and EPR can serve as an indicator of MOF crystal packing quality/density when biomineralized in MOFs. The method can be generalized to probing the dynamics of other enzymes on other solid surfaces/interfaces and guide the rational design of solid platforms (ca. MOFs) to customize enzyme immobilization.

Biobased, Macro-, and Nanoscale Fungicide Delivery Approaches for Plant Fungi Control.

In this report, two polymeric matrix systems at macro and nanoscales were prepared for efficacious fungicide delivery. The macroscale delivery systems used millimeter-scale, spherical beads composed of cellulose nanocrystals and poly(lactic acid). The nanoscale delivery system involved micelle-type nanoparticles, composed of methoxylated sucrose soyate polyols. Sclerotinia sclerotiorum (Lib.), a destructive fungus affecting high-value industrial crops, was used as a model pathogen against which the efficacy of these polymeric formulations was demonstrated. Commercial fungicides are applied on plants frequently to overcome the transmission of fungal infection. However, fungicides alone do not persist on the plants for a prolonged period due to environmental factors such as rain and airflow. There is a need to apply fungicides multiple times. As such, standard application practices generate a significant environmental footprint due to fungicide accumulation in soil and runoff in surface water. Thus, approaches are needed that can either increase the efficacy of commercially active fungicides or prolong their residence time on plants for sustained antifungal coverage. Using azoxystrobin (AZ) as a model fungicide and canola as a model crop host, we hypothesized that the AZ-loaded macroscale beads, when placed in contact with plants, will act as a depot to release the fungicide at a controlled rate to protect plants against fungal infection. The nanoparticle-based fungicide delivery approach, on the other hand, can be realized via spray or foliar applications. The release rate of AZ from macro- and nanoscale systems was evaluated and analyzed using different kinetic models to understand the mechanism of AZ delivery. We observed that, for macroscopic beads, porosity, tortuosity, and surface roughness governed the efficiency of AZ delivery, and for nanoparticles, contact angle and surface adhesion energy were directing the efficacy of the encapsulated fungicide. The technology reported here can also be translated to a wide variety of industrial crops for fungal protection. The strength of this study is the possibility of using completely plant-derived, biodegradable/compostable additive materials for controlled agrochemical delivery formulations, which will contribute to reducing the frequency of fungicide applications and the potential accumulation of formulation components in soil and water.

Cutting Boards: An Overlooked Source of Microplastics in Human Food?

Plastic cutting boards are a potentially significant source of microplastics in human food. Thus, we investigated the impact of chopping styles and board materials on microplastics released during chopping. As chopping progressed, the effects of chopping styles on microplastic release became evident. The mass and number of microplastics released from polypropylene chopping boards were greater than polyethylene by 5-60% and 14-71%, respectively. Chopping on polyethylene boards was associated with a greater release of microplastics with a vegetable (i.e., carrots) than chopping without carrots. Microplastics showed a broad, bottom-skewed normal distribution, dominated by <100 µm spherical-shaped microplastics. Based on our assumptions, we estimated a per-person annual exposure of 7.4-50.7 g of microplastics from a polyethylene chopping board and 49.5 g of microplastics from a polypropylene chopping board. We further estimated that a person could be exposed to 14.5 to 71.9 million polyethylene microplastics annually, compared to 79.4 million polypropylene microplastics from chopping boards. The preliminary toxicity study of the polyethylene microplastics did not show adverse effects on the viability of mouse fibroblast cells for 72 h. This study identifies plastic chopping boards as a substantial source of microplastics in human food, which requires careful attention.

The role of CCNs in controlling cellular communication in the tumor microenvironment.

The Cellular communication network (CCN) family of growth regulatory factors comprises six secreted matricellular proteins that promote signal transduction through cell-cell or cell-matrix interaction. The diversity of functionality between each protein is specific to the many aspects of healthy and cancer biology. For example, CCN family proteins modulate cell adhesion, proliferation, migration, invasiveness, apoptosis, and survival. In addition, the expression of each protein regulates many biological and pathobiological processes within its microenvironment to regulate angiogenesis, inflammatory response, chondrogenesis, fibrosis, and mitochondrial integrity. The collective range of CCN operation remains fully comprehended; however, understanding each protein's microenvironment may draw more conclusions about the abundance of interactions and signaling cascades occurring within such issues. This review observes and distinguishes the various roles a CCN protein may execute within distinct tumor microenvironments and the biological associations among them. Finally. We also review how CCN-family proteins can be used in nano-based therapeutic implications.

Bioinspired Materials for Wearable Devices and Point-of-Care Testing of Cancer.

Wearable, point-of-care diagnostics, and biosensors are on the verge of bringing transformative changes in detection, management, and treatment of cancer. Bioinspired materials with new forms and functions have frequently been used, in both translational and commercial spaces, to fabricate such diagnostic platforms. Engineered from organic or inorganic molecules, bioinspired systems are naturally equipped with biorecognition and stimuli-sensitive properties. Mechanisms of action of bioinspired materials are deeply connected with thermodynamically or kinetically controlled self-assembly at the molecular and supramolecular levels. Thus, integration of bioinspired materials into wearable devices, either as triggers or sensors, brings about unique device properties usable for detection, capture, or rapid readout for an analyte of interest. In this review, we present the basic principles and mechanisms of action of diagnostic devices engineered from bioinspired materials, describe current advances, and discuss future trends of the field, particularly in the context of cancer.

Nano Boron Oxide and Zinc Oxide Doped Lignin Containing Cellulose Nanocrystals Improve the Thermal, Mechanical and Flammability Properties of High-Density Poly(ethylene).

The widely used high-density polyethylene (HDPE) polymer has inadequate mechanical and thermal properties for structural applications. To overcome this challenge, nano zinc oxide (ZnO) and nano boron oxide (B2O3) doped lignin-containing cellulose nanocrystals (L-CNC) were blended in the polymer matrix. The working hypothesis is that lignin will prevent CNC aggregation, and metal oxides will reduce the flammability of polymers by modifying their degradation pathways. This research prepared and incorporated safe, effective, and eco-friendly hybrid systems of nano ZnO/L-CNC and nano B2O3/L-CNC into the HDPE matrix to improve their physio-mechanical and fire-retardant properties. The composites were characterized using Fourier transform infrared spectroscopy, scanning electron microscopy, energy dispersive X-ray analysis, thermo-gravimetric analysis, differential scanning calorimetry, dynamic mechanical analysis, horizontal burning test, and microcalorimetry test. The results demonstrated a substantial increase in mechanical properties and a reduction in flammability. The scanning electron microscope (SEM) images showed some agglomeration and irregular distribution of the inorganic oxides.

Expanding the "Library" of Metal-Organic Frameworks for Enzyme Biomineralization.

Metal-organic frameworks (MOFs) are advanced platforms for enzyme immobilization. Enzymes can be entrapped via either diffusion (into pre-formed MOFs) or co-crystallization. Enzyme co-crystallization with specific metals/ligands in the aqueous phase, also known as biomineralization, minimizes the enzyme loss compared to organic phase co-crystallization, removes the size limitation on enzymes and substrates, and can potentially broaden the application of enzyme@MOF composites. However, not all enzymes are stable/functional in the presence of excess metal ions and/or ligands currently available for co-crystallization. Furthermore, most current biomineralization-based MOFs have limited (acid) pH stability, making it necessary to explore other metal-ligand combinations that can also immobilize enzymes. Here, we report our discovery on the combination of five metal ions and two ligands that can form biocomposites with two model enzymes differing in size and hydrophobicity in the aqueous phase under ambient conditions. Surprisingly, most of the formed composites are single- or multiphase crystals, even though the reaction phase is aqueous, with the rest as amorphous powders. All 20 enzyme@MOF composites showed good to excellent reusability and were stable under weakly acidic pH values. The stability under weakly basic conditions depended upon the selection of enzyme and metal-ligand combinations, yet for both enzymes, 3-4 MOFs offered decent stability under basic conditions. This work initiates the expansion of the current "library" of metal-ligand selection for encapsulating/biomineralizing large enzymes/enzyme clusters, leading to customized encapsulation of enzymes according to enzyme stability, functionality, and optimal pH.

Cellulose nanofibers as Scaffold-forming materials for thin film drug delivery systems.

We explored the potential of cellulose nanofiber (CNF) for designing prolonged-release, thin-film drug delivery systems (TF-DDS). These delivery systems can be used as locally deployable drug-releasing scaffolds for achieving spatial and temporal control over therapeutic concentration in target tissues. Using doxorubicin (DOX) as a model anticancer drug, CNF-based TF-DDS were prepared using different film-formation processes, such as solvent casting and lyophilization. Formulations were prepared with or without the incorporation of additional macromolecular additives, such as gelatin, to include further biomechanical functionality. We studied the films for their mechanical properties, thermal stability, wettability, porosity and in vitro drug release properties. Our experimental results showed that CNF-based films, when prepared via solvent casting method, showed optimized performance in terms of DOX loading, and prolonged-release than those prepared via lyophilization-based fabrication processes. Scanning electron microscopy (SEM) analysis of the CNF-based films showed uniform distribution of fiber entanglement, which provided the scaffolds with sufficient porosity and tortuosity contributing to the sustained release of the drug from the delivery system. We also observed that surface layering of gelatin on CNF films via dip-coating significantly increased the mechanical strength and reduced the wettability of the films, and as such, affected drug release kinetics. The performance of the TF-DDS was evaluated in-vitro against two pancreatic cancer cell lines, i.e. MIA PaCa-2 and PANC-1. We observed that, along with the enhancement of mean dissolution time (MDT) of DOX, CNF-based TF-DDS were able to suppress the proliferation of pancreatic cancer cells in a time-dependent fashion, indicating that the drug liberated from the films were therapeutically active against cancer cells. Additionally, TF-DDS were also tested ex-vivo on patient-derived xenograft (PDX) model of pancreatic ductal adenocarcinoma (PDAC). We observed that DOX released from the TF-DDS was able to reduce Ki-67 positive, pancreatic cancer cells in these models.

Self-Assembled Nanostructures from Amphiphilic Sucrose-Soyates for Solubilizing Hydrophobic Guest Molecules.

We studied self-assembly and colloidal properties of poly(ethylene glycol) (pEG) conjugated sucrose soyate polyols (PSSP). These molecular platforms were synthesized by covalently connecting PEGs of different molecular weights (Mn) (12 and 16 ethylene oxide units) to epoxidized sucrose soyate (ESS). The synthesized PSSP products showed amphiphilicity, reduced water surface tension, and exhibited critical Aggregation Concentration (CAC) within the range of 0.3-0.4 mg/mL. We observed that PSSP self-assembles in water in the form of nanoparticles without the need of any cosolvents. These nanoparticles exhibited number-average hydrodynamic diameter of 120 ± 8 nm with a polydispersity index (PDI) of <0.3, and negatively charged surfaces. We also found out that PSSP nanoparticles can encapsulate and homogeneously distribute a hydrophobic model compound, such as a phthalocyanine dye, Solvent Blue-70 (BL-70), on a metal surface. Collectively, our studies explored and demonstrated the possibility of molecular diversification of biobased starting materials to form amphiphilic nanoparticles with industrially relevant colloidal and surface properties.

Polymeric Composite Matrix with High Biobased Content as Pharmaceutically Relevant Molecular Encapsulation and Release Platform.

Drug delivery systems (DDS) that can temporally control the rate and extent of release of therapeutically active molecules find applications in many clinical settings, ranging from infection control to cancer therapy. With an aim to design a locally implantable, controlled-release DDS, we demonstrated the feasibility of using cellulose nanocrystal (CNC)-reinforced poly (l-lactic acid) (PLA) composite beads. The performance of the platform was evaluated using doxorubicin (DOX) as a model drug for applications in triple-negative breast cancer. A facile, nonsolvent-induced phase separation (NIPS) method was adopted to form composite beads. We observed that CNC loading within these beads played a critical role in the mechanical stability, porosity, water uptake, diffusion, release, and pharmacological activity of the drug from the delivery system. When loaded with DOX, composite beads significantly controlled the release of the drug in a pH-dependent pattern. For example, PLA/CNC beads containing 37.5 wt % of CNCs showed a biphasic release of DOX, where 41 and 82% of the loaded drug were released at pH 7.4 and pH 5.5, respectively, over 7 days. Drug release followed Korsmeyer's kinetics, indicating that the release mechanism was mostly diffusion and swelling-controlled. We showed that DOX released from drug-loaded PLA/CNC composite beads locally suppressed the growth and proliferation of triple-negative breast cancer cells, MBA-MB-231, via the apoptotic pathway. The efficacy of the DDS was evaluated in human tissue explants. We envision that such systems will find applications for designing biobased platforms with programmed stability and drug delivery functions.

CCN5 activation by free or encapsulated EGCG is required to render triple-negative breast cancer cell viability and tumor progression.

Epigallocatechin-3-gallate (EGCG) has been considered an anticancer agent despite conflicting and discrepant bioavailability views. EGCG impairs the viability and self-renewal capacity of triple-negative breast cancer (TNBC) cells and makes them sensitive to estrogen via activating ER-α. Surprisingly, the mechanism of EGCG's action on TNBC cells remains unclear. CCN5/WISP-2 is a gatekeeper gene that regulates viability, ER-α, and stemness in TNBC and other types of cancers. This study aimed to investigate whether EGCG (free or encapsulated in nanoparticles) interacts with the CCN5 protein by emphasizing its bioavailability and enhancing its anticancer effect. We demonstrate that EGCG activates CCN5 to inhibit in vitro cell viability through apoptosis, the sphere-forming ability via reversing TNBC cells' stemness, and suppressing tumor growth in vivo. Moreover, we found EGCG-loaded nanoparticles to be functionally more active and superior in their tumor-suppressing ability than free-EGCG. Together, these studies identify EGCG (free or encapsulated) as a novel activator of CCN5 in TNBC cells and hold promise as a future therapeutic option for TNBC with upregulated CCN5 expression.

Chemo-specific designs for the enumeration of circulating tumor cells: advances in liquid biopsy.

Advanced materials and chemo-specific designs at the nano/micrometer-scale have ensured revolutionary progress in next-generation clinically relevant technologies. For example, isolating a rare population of cells, like circulating tumor cells (CTCs) from the blood amongst billions of other blood cells, is one of the most complex scientific challenges in cancer diagnostics. The chemical tunability for achieving this degree of exceptional specificity for extra-cellular biomarker interactions demands the utility of advanced entities and multistep reactions both in solution and in the insoluble state. Thus, this review delineates the chemo-specific substrates, chemical methods, and structure-activity relationships (SARs) of chemical platforms used for isolation and enumeration of CTCs in advancing the relevance of liquid biopsy in cancer diagnostics and disease management. We highlight the synthesis of cell-specific, tumor biomarker-based, chemo-specific substrates utilizing functionalized linkers through chemistry-based conjugation strategies. The capacity of these nano/micro substrates to enhance the cell interaction specificity and efficiency with the targeted tumor cells is detailed. Furthermore, this review accounts for the importance of CTC capture and other downstream processes involving genotypic and phenotypic CTC analysis in real-time for the detection of the early onset of metastases progression and chemotherapy treatment response, and for monitoring progression free-survival (PFS), disease-free survival (DFS), and eventually overall survival (OS) in cancer patients.

New side chain design for pH-responsive block copolymers for drug delivery.

New molecular motifs that can act as pH-regulating triggers for amphiphilic, pH-sensitive block copolymers are investigated. Inspired by the mechanism of action of pH-indicators, such as methyl orange, and natural amino acids, we designed these copolymers where either 4-Amino-4'-dimethylaminoazobenzene, AZB (pKa 3.4, an amine derivative of methyl orange), isoleucine, Ile (pKa 2.37 for carboxylic acid), or a statistical mixture of both were appended as side chains to the hydrophobic block to act as pH-triggers. These new side chain motifs were identified with an aim to enhance the self-assembling properties of the block copolymers in terms of particle size and stability, drug encapsulation, and release. As the parent polymer, poly (ethylene) glycol-block- poly (carbonate) (PEG-b-PC) of number average molecular weight 12.1 kDa was used. We observed that PEG-b-PC block copolymers, when engineered with AZB or Ile-type of pH-regulators appended as side chains to PC blocks, formed self-assembled, spherical nanoparticles with hydrodynamic diameters ranging from 114 to 137 nm depending on copolymer composition. Critical aggregation concentrations (CAC) of the block copolymers were found to be governed by the type and content of side chains. We explored the use of these newly designed block copolymer assemblies as drug carriers using gemcitabine (GEM) as a model cytotoxic drug generally used for pancreatic ductal adenocarcinoma (PDAC). We showed that AZB and Ile decorated copolymeric nanocarriers were able to encapsulate GEM at 13.8-28.8 % loading content and release the drug in a pH-dependent pattern. Drug-loaded nanocarriers showed cellular entry into PDAC cells in vitro and were found to exert cytotoxicity against these cells. Neither the block copolymers bearing AZB or Ile-type pH-responsive triggers, nor their self-assembled nanoparticles showed any cytotoxicity at usable concentrations, thereby reflecting the potentials of these molecular motifs for designing stimuli-responsive drug delivery nanosystems.

pH-Sensitive Nanodrug Carriers for Codelivery of ERK Inhibitor and Gemcitabine Enhance the Inhibition of Tumor Growth in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC), a metabolic disorder, remains one of the leading cancer mortality sources worldwide. An initial response to treatments, such as gemcitabine (GEM), is often followed by emergent resistance reflecting an urgent need for alternate therapies. The PDAC resistance to GEM could be due to ERK1/2 activity. However, successful ERKi therapy is hindered due to low ligand efficiency, poor drug delivery, and toxicity. In this study, to overcome these limitations, we have designed pH-responsive nanoparticles (pHNPs) with a size range of 100-150 nm for the simultaneous delivery of ERKi (SCH 772984) and GEM with tolerable doses. These pHNPs are polyethylene glycol (PEG)-containing amphiphilic polycarbonate block copolymers with tertiary amine side chains. They are systemically stable and capable of improving in vitro and in vivo drug delivery at the cellular environment's acidic pH. The functional analysis indicates that the nanomolar doses of ERKi or GEM significantly decreased the 50% growth inhibition (IC50) of PDAC cells when encapsulated in pHNPs compared to free drugs. The combination of ERKi with GEM displayed a synergistic inhibitory effect. Unexpectedly, we uncover that the minimum effective dose of ERKi significantly promotes GEM activities on PDAC cells. Furthermore, we found that pHNP-encapsulated combination therapy of ERKi with GEM was superior to unencapsulated combination drug therapy. Our findings, thus, reveal a simple, yet efficient, drug delivery approach to overcome the limitations of ERKi for clinical applications and present a new model of sensitization of GEM by ERKi with no or minimal toxicity.

Chemical Architecture of Block Copolymers Differentially Abrogate Cardiotoxicity and Maintain the Anticancer Efficacy of Doxorubicin.

The molecular architecture of pH-responsive amphiphilic block copolymers, their self-assembly behavior to form nanoparticles (NPs), and doxorubicin (DOX)-loading technique govern the extent of DOX-induced cardiotoxicity. We observed that the choice of pH-sensitive tertiary amines, surface charge, and DOX-loading techniques within the self-assembled NPs strongly influence the release and stimulation of DOX-induced cardiotoxicity in primary cardiomyocytes. However, covalent conjugation of DOX to a pH-sensitive nanocarrier through a "conditionally unstable amide" linkage (PCPY-cDOX; PC = polycarbonate and PY = 2-pyrrolidine-1-yl-ethyl-amine) significantly reduced the cardiotoxicity of DOX in cardiomyocytes as compared to noncovalently encapsulated DOX NPs (PCPY-eDOX). When these formulations were tested for drug release in serum-containing media, the PCPY-cDOX systems showed prolonged control over drug release (for ∼72 h) at acidic pH compared to DOX-encapsulated nanocarriers, as expected. We found that DOX-encapsulated nanoformulations triggered cardiotoxicity in primary cardiomyocytes more acutely, while conjugated systems such as PCPY-cDOX prevented cardiotoxicity by disabling the nuclear entry of the drug. Using 2D and 3D (spheroid) cultures of an ER + breast cancer cell line (MCF-7) and a triple-negative breast cancer cell line (MDA-MB-231), we unravel that, similar to encapsulated systems (PCPY-eDOX-type) as reported earlier, the PCPY-cDOX system suppresses cellular proliferation in both cell lines and enhances trafficking through 3D spheroids of MDA-MB-231 cells. Collectively, our studies indicate that PCPY-cDOX is less cardiotoxic as compared to noncovalently encapsulated variants without compromising the chemotherapeutic properties of the drug. Thus, our studies suggest that the appropriate selection of the nanocarrier for DOX delivery may prove fruitful in shifting the balance between low cardiotoxicity and triggering the chemotherapeutic potency of DOX.

Author Correction: Cellulose Mediated Transferrin Nanocages for Enumeration of Circulating Tumor Cells for Head and Neck Cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.