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.

Although enzyme immobilization has improved many areas, biocatalysis involving large-size substrates is still challenging for immobilization platform design because of the protein damage under the often “harsh” reaction conditions required for these reactions. Our recent efforts indicate the potential of using Metal–Organic Frameworks (MOFs) to partially confine enzymes on the surface of MOF-based composites while offering sufficient substrate contact. Still, improvements are required to expand the feasible pH range and the efficiency of contacting substrates. In this contribution, we discovered that Zeolitic Imidazolate Framework (ZIF) and a new calcium-carboxylate based MOF (CaBDC) can both be coprecipitated with a model large-substrate enzyme, lysozyme (lys), to anchor the enzyme on the surface of graphite oxide (GO). We observed lys activity against its native substrate, bacterial cell walls, indicating lys was confined on composite surface. Remarkably, lys@GO/CaBDC displayed a stronger catalytic efficiency at pH 6.2 as compared to pH 7.4, indicating CaBDC is a good candidate for biocatalysis under acidic conditions as compared to ZIFs which disassemble under pH < 7. Furthermore, to understand the regions of lys being exposed to the reaction medium, we carried out a site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy study. Our data showed a preferential orientation of lys in GO/ZIF composite, whereas a random orientation in GO/CaBDC. This is the first report on immobilizing solution-state large-substrate enzymes on GO surface using two different MOFs via one-pot synthesis. These platforms can be generalized to other large-substrate enzymes to carry out catalysis under the optimal buffer/pH conditions. The orientation of enzyme at the molecular level on composite surfaces is critical for guiding the rational design of new composites.

Immobilizing enzymes on nanoparticles (NPs) enhances the cost-efficiency of biocatalysis; however, when the substrates are large, it becomes difficult to separate the enzyme@NP from the products while avoiding leaching or damage of enzymes in the reaction medium. Metal–organic framework (MOF)-coated magnetic NPs (MNPs) offer efficient magnetic separation and enhanced enzyme protection; however, the involved enzymes/substrates have to be smaller than the MOF apertures. A potential solution to these challenges is coprecipitating metal/ligand with enzymes on the MNP surface, which can partially bury (protect) the enzyme below the composite surface while exposing the rest of the enzyme to the reaction medium for catalysis of larger substrates. Here, to prove this concept, we show that using Ca2+ and terephthalic acid (BDC), large-substrate enzymes can be encapsulated in CaBDC-MOF layers coated on MNPs via an enzyme-friendly, aqueous-phase one-pot synthesis. Interestingly, we found that using MNPs as the nuclei of crystallization, the composite size can be tuned so that nanoscale composites were formed under low Ca2+/BDC concentrations, while microscale composites were formed under high Ca2+/BDC concentrations. While the microscale composites showed significantly enhanced reusability against a non-structured large substrate, the nanoscale composites displayed enhanced catalytic efficiency against a rigid, crystalline-like large substrate, starch, likely due to the improved diffusivity of the nanoscale composites. To our best knowledge, this is the first report on aqueous-phase one-pot synthesis of size-tunable enzyme@MOF/MNP composites for large-substrate biocatalysis. Our platform can be applied to immobilize other large-substrate enzymes with enhanced reusability and tunable sizes.

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 αvβ3 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.

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.