Cholesterol lowering in diet-induced hypercholesterolemic mice using Lactobacillus bile salt hydrolases with different substrate specificities.

The cholesterol-lowering effect of lactic acid bacteria with high activity of bile salt hydrolase (BSH) is unclear. We believe that distinguishing BSH substrate specificity is necessary to study the effect of various BSH enzymes. We engineered a BSH mutant enzyme recombinant strain named F67A, which exclusively hydrolyzes taurocholic acid (TCA) using site-directed mutagenesis, and a previously lab-constructed BSH recombinant strain, YB81 that exclusively hydrolyzes glycocholic acid (GCA). We also constructed the recombinant strain named NB5462, which carries the empty pSIP411 plasmid and was used as a blank control strain. The intestinal flora in pseudo-germ-free (PGF) mice in which intestinal flora were eliminated via antibiotics, and F67A successfully reduced serum cholesterol levels in high-cholesterol diet-fed mice, whereas YB81 did not yield the same results. However, YB81 regained its cholesterol-lowering capacity in specific pathogen-free (SPF) mice with intact intestinal flora. The cholesterol-lowering mechanism of F67A involved modifying the bile acid pool through BSH enzyme activity. This adjustment regulated the expression of intestinal farnesoid X receptor and subsequently elevated hepatic cholesterol 7α-hydroxylase (CYP7A1), effectively reducing cholesterol levels. Conversely, GCA, the substrate of YB81, was found in minimal quantities in mice, preventing it from inducing changes in bile acid pools. In the presence of intestinal flora, the YB81 BSH enzyme induced notable alterations in bile acids by regulating changes in the intestinal flora and BSH within the flora, ultimately resulting in cholesterol reduction. This is the first study investigating the substrate specificity of BSH, demonstrating that different substrate-specific BSH enzymes exhibit cholesterol-lowering properties. Additionally, we elaborate on the mechanism of BSH-mediated enterohepatic axis regulation.

Molecularly Imprinted Nanomedicine for Anti-angiogenic Cancer Therapy via Blocking Vascular Endothelial Growth Factor Signaling.

The VEGF-VEGFR2 (VEGF = vascular endothelial growth factor) signaling has been a promising target in cancer therapy. However, because conventional anti-angiogenic therapeutics suffer from drawbacks, particularly severe side effects, novel anti-angiogenic strategies are much needed. Herein, we report the rational engineering of VEGF-targeted molecularly imprinted polymer nanoparticles (nanoMIP) for anti-angiogenic cancer therapy. The anti-VEGF nanomedicine was prepared via a state-of-the-art molecular imprinting approach using the N-terminal epitope of VEGF as the template. The nanoMIP could target the two major pro-angiogenic isoforms (VEGF165 and VEGF121) with high affinity and thereby effectively block the VEGF-VEGFR2 signaling, yielding a potent anti-angiogenic effect of "killing two birds with one stone". In vivo experiments demonstrated that the anti-VEGF nanoMIP effectively suppressed tumor growth via anti-angiogenesis in a xenograft model of human colon carcinoma without apparent side effects. Thus, this study not only proposes an unprecedented anti-angiogenic strategy for cancer therapy but also provides a new paradigm for the rational development of MIPs-based "drug-free" nanomedicines.

Photothermal Therapy of Neuroblastoma via Polysialic Acid-Targeting Nanomissiles.

Exploring new targets and developing novel targeted therapies are urgently needed for neuroblastoma therapy. Polysialic acid (polySia), a linear homopolymer of sialic acid units that correlates well with tumor progression and poor prognosis, has emerged as a potential target for neuroblastoma. However, the lack of polySia-specific binding reagents has severely limited the development of polySia-targeting therapeutics for neuroblastoma. Herein, the construction of polySia-targeting nanomissiles via molecular imprinting for the photothermal therapy of neuroblastoma is reported. Oligosialic acid (oligoSia) containing 3-4 units is considered as a characteristic structure for the recognition of polySia, while oligoSia containing 4-7 units digested from polySia is employed as the template. Via boronate-affinity controllable oriented surface imprinting, oligoSia-imprinted nanoparticles (oSia-MIP) are prepared. The oSia-MIP allows for specifically recognizing polySia and targeting polySia overexpressed neuroblastoma cells in vitro and in vivo. oSia-MIP loaded with indocyanine green is prepared and experimentally demonstrated to be a potent targeted photothermal therapeutic for neuroblastoma. Equipping the core substrate with functional entities, the developed polySia targeting nanoplatform can be accommodated to various therapeutic modalities, holding great promise for neuroblastoma targeted therapy.

Boosting Chemodynamic Therapy by Tumor-Targeting and Cellular Redox Homeostasis-Disrupting Nanoparticles.

Chemodynamic therapy (CDT) that kills tumor cells by converting low-reactivity H2O2 into highly toxic hydroxyl radicals (•OH) is an emerging tumor therapeutic modality, but its therapeutic efficacy is largely limited by both the lack of tumor targeting and redox homeostasis in tumor cells. Herein, we report Cu2+-encapsulated and GalNAc-imprinted biodegradable silica nanoparticles (nanoMIP) for boosting CDT. In this strategy, the Cu2+ was first encapsulated into disulfide-bridged silica nanoparticles with a high loading capacity of ∼18.3%, followed by in situ functionalization via molecular imprinting using GalNAc as a template. Such a nanovector could specifically target tumor cells overexpressing the Tn antigen to promote the cellular uptake. After internalization into tumor cells, the degradation of nanoMIP occurred in response to the tumor microenvironment, spontaneously releasing Cu2+/Cu+ via redox cycles, which in turn promoted highly potent GSH depletion and triggered •OH generation by a Fenton-like reaction. Notably, we found that the catalase activity could be effectively inhibited by the produced Cu+, which indirectly upregulated the endogenous H2O2 level. As a result, the "maladjusted" tumor cells lost the resistance against •OH damage, finally resulting in the apoptosis of tumor cells. In vitro and in vivo experiments demonstrated that our nanoMIP exhibited excellent cytotoxicity against tumor cells and high efficacy of tumor inhibition in the xenograft tumor model with negligible side effects. Taken together, our study provides not only a promising strategy for maximizing the CDT efficacy but also a new insight for developing MIP-based nanomedicine.

Molecularly imprinted and cladded nanoparticles for high-affinity recognition of structurally closed gangliosides.

A new method called reverse microemulsion-confined ganglioside-oriented surface imprinting and cladding (RM-GOSIC) is presented for controllable preparation of nanoscale binders for high-affinity targeting gangliosides. Using GM1a, an affordable ganglioside, as a representative ganglioside target, single-core quantum dot GM1a-imprinted and GM1a-cladded polymer (cMIP) nanoparticles were prepared. The prepared cMIP nanoparticles exhibited extremely high affinity towards GM1a, with dissociation constant at the nanomolar level (3-6 nM). The prepared cMIP nanoparticles also recognized structurally closed gangliosides while their cross-reactivity towards other gangliosides remained low. The potential of the cMIP nanoparticles in biomedical applications was demonstrated by cell and tissue imaging. Thus, this approach opened a new access to the synthesis of high-affinity nanoscale binders for targeting gangliosides.

Controllable Engineering and Functionalizing of Nanoparticles for Targeting Specific Proteins towards Biomedical Applications.

Nanoparticles have been widely used in important biomedical applications such as imaging, drug delivery, and disease therapy, in which targeting toward specific proteins is often essential. However, current targeting strategies mainly rely on surface modification with bioligands, which not only often fail to provide desired properties but also remain challenging. Here an unprecedented approach is reported, called reverse microemulsion-confined epitope-oriented surface imprinting and cladding (ROSIC), for facile, versatile, and controllable engineering coreless and core/shell nanoparticles with tunable monodispersed size as well as specific targeting capability toward proteins and peptides. Via engineering coreless imprinted and cladded silica nanoparticles, the effectiveness and superiority over conventional imprinting of the proposed approach are first verified. The prepared nanoparticles exhibit both high specificity and high affinity. Using quantum dots, superparamagnetic nanoparticles, silver nanoparticles, and upconverting nanoparticles as a representative set of core substrates, a variety of imprinted and cladded single-core/shell nanoparticles are then successfully prepared. Finally, using imprinted and cladded fluorescent nanoparticles as probes, in vitro targeted imaging of triple-negative breast cancer (TNBC) cells and in vivo targeted imaging of TNBC-bearing mice are achieved. This approach opens a new avenue to engineering of nanoparticles for targeting specific proteins, holding great prospects in biomedical applications.

Redox-Responsive Molecularly Imprinted Nanoparticles for Targeted Intracellular Delivery of Protein toward Cancer Therapy.

Although protein therapeutics is of significance in therapeutic intervention of cancers, controlled delivery of therapeutic proteins still faces substantial challenges including susceptibility to degradation and denaturation and poor membrane permeability. Herein, we report a sialic acid (SA)-imprinted biodegradable silica nanoparticles (BS-NPs)-based protein delivery strategy for targeted cancer therapy. Cytotoxic ribonuclease A (RNase A) was effectively caged in the matrix of disulfide-hybridized silica NPs (encapsulation efficiency of ∼64%), which were further functionalized with cancer targeting capability via surface imprinting with SA as imprinting template. Such nanovectors could not only maintain high stability in physiological conditions but also permit redox-triggered biodegradation for both concomitant release of the loaded therapeutic cargo and in vivo clearance. In vitro experiments confirmed that the SA-imprinted RNase A@BS-NPs could selectively target SA-overexpressed tumor cells, promote cells uptake, and subsequently be cleaved by intracellular glutathione (GSH), resulting in rapid release kinetics and enhanced cell cytotoxicity. In vivo experiments further confirmed that the SA-imprinted RNase A@BS-NPs had specific tumor-targeting ability and high therapeutic efficacy of RNase A in xenograft tumor model. Due to the specific targeting and traceless GSH-stimulated intracellular protein release, the SA-imprinted BS-NPs provided a promising platform for the delivery of biomacromolecules in cancer therapy.