Enzyme-responsive Nanoparticles Research Articles

Enzyme-responsive nanoparticles: enhancing the ability of endolysins to eradicate Staphylococcus aureus biofilm.

Stimuli-responsive nanomaterials show promise in eradicating Staphylococcus aureus biofilm from implants. Peptidoglycan hydrolases (PGHs) are cationic antimicrobials that can be bioengineered to improve the targeting of persisters and drug-resistant bacteria. However, these molecules can be degraded before reaching the target and/or present limited efficacy against biofilm. Therefore, there is an urgent need to improve their potency. Herein, PGH-polyphosphate nanoparticles (PGH-PP NPs) are formed by ionotropic gelation between cationic PGHs and anionic polyphosphate, with the aim of protecting PHGs and delivering them at the target site triggered by alkaline phosphatase (AP) from S. aureus biofilm. Optimized conditions for obtaining M23-PP NPs and GH15-PP NPs are presented. Size, zeta potential, and transmission electron microscopy imaging confirm the nanoscale size. The system demonstrates outstanding performance, as evidenced by a dramatic reduction in PGHs' minimum inhibitory concentration and minimum bactericidal concentration, together with protection against proteolytic effects, storage stability, and cytotoxicity towards the Caco-2 and HeLa cell lines. Time-kill experiments show the great potential of these negatively charged delivery systems in overcoming the staphylococcal biofilm barrier. Efficacy under conditions inhibiting AP proves the enzyme-triggered delivery of PGHs. The enzyme-responsive PGH-PP NPs significantly enhance the effectiveness of PGHs against bacteria residing in biofilm, offering a promising strategy for eradicating S. aureus biofilm.

Immunosuppressive enzyme-responsive nanoparticles for enhanced accumulation in liver allograft to overcome acute rejection

Acute rejection is a life-threatening complication after liver transplantation. Immunosuppressants such as tacrolimus are used to inhibit acute rejection of liver grafts in clinic. However, inefficient intragraft accumulation may reduce the therapeutic outcomes of tacrolimus. Here, an enzyme-responsive nanoparticle is developed to selectively enhance the accumulation of tacrolimus in liver allograft through enzyme-induced aggregation to refine immunotherapeutic efficacy of tacrolimus. The nanoparticles are composed of amphiphilic tacrolimus prodrugs synthesized by covalently conjugating tacrolimus and matrix metalloproteinase 9 (MMP9)-cleavable peptide-containing methoxy poly (ethylene glycol) to poly (l-glutamic acid). Upon exposure to MMP9, which is overexpressed in rejected liver allografts, the nanoparticles undergo a morphological transition from spherical micellar nanoparticles to microscale aggregate-like scaffolds. Intravenous administration of MMP9-responsive nanoparticles into a rat model of acute liver graft rejection results in enhanced nanoparticle accumulation in allograft as compared to nonresponsive nanoparticles. Consequently, the MMP9-responsive nanoparticles significantly inhibit intragraft inflammatory cell infiltration and proliferation, maintain intragraft immunosuppressive environment, alleviate graft damage, improve liver allograft function, abate weight loss and prolong recipient survival. This work proves that morphology-switchable enzyme-responsive nanoparticles represent an innovative strategy for selectively enhancing intragraft accumulation of immunosuppressive agents to improve treatment of liver allograft rejection.

Enzyme-Responsive Nanoparticles for the Targeted Delivery of an MMP Inhibitor to Acute Myocardial Infarction.

Herein, we have developed a drug-loaded matrix metalloproteinase (MMP)-responsive micellar nanoparticle (NP) intended for minimally invasive intravenous injection during the acute phase of myocardial infarction (MI) and prolonged retention in the heart for small-molecule drug delivery. Peptide-polymer amphiphiles (PPAs) bearing a small-molecule MMP inhibitor (MMPi), PD166793, were synthesized via ring-opening metathesis polymerization (ROMP) and formulated into spherical micelles by transitioning to aqueous solution. The resulting micellar NPs underwent MMP-induced aggregation, demonstrating enzyme responsiveness. Using a rat MI model, we observed that these NPs were capable of successfully extravasating into the infarcted region of the heart where they were retained due to the active, enzyme-mediated targeting, remaining detectable after 1 week post administration without increasing macrophage recruitment. Furthermore, in vitro studies show that these NPs demonstrated successful drug release following MMP treatment and maintained drug bioactivity as evidenced by comparable MMP inhibition to free MMPi. This work establishes a targeted NP platform for delivering small-molecule therapeutics to the heart after MI, opening possibilities for myocardial infarction treatment.

Enzyme-Responsive Nanoparticles for Dexamethasone Targeted Delivery to Treat Inflammation in Diabetes.

Diabetes is a global epidemic accompanied by impaired wound healing and increased risk of persistent infections and resistance to standard treatments. Therefore, there is an immense need to develop novel methods to specifically target therapeutics to affected tissues and improve treatment efficacy. This study aims to use enzyme-responsive nanoparticles for the targeted delivery of an anti-inflammatory drug, dexamethasone, to treat inflammation in diabetes. These nanoparticles are assembled from fluorescently-labeled, dexamethasone-loaded peptide-polymer amphiphiles. The nanoparticles are injected in vivo, adjacent to labeled collagen membranes sub-periosteally implanted on the calvaria of diabetic rats. Following their implantation, collagen membrane resorption is linked to inflammation, especially in hyperglycemic individuals. The nanoparticles show strong and prolonged accumulation in inflamed tissue after undergoing a morphological switch into microscale aggregates. Significantly higher remaining collagen membrane area and less inflammatory cell infiltration are observed in responsive nanoparticles-treated rats, compared to control groups injected with free dexamethasone and non-responsive nanoparticles. These factors indicate improved therapeutic efficacy in inflammation reduction. These results demonstrate the potential use of enzyme-responsive nanoparticles as targeted delivery vehicles for the treatment of diabetic and other inflammatory wounds.

Enhancing Anti-Tumorigenic Efficacy of Eugenol in Human Colon Cancer Cells Using Enzyme-Responsive Nanoparticles

This study is focused on the selective delivery and release of the plant-based anticancer compound eugenol (EUG) in colorectal cancer cells (CRC). EUG is an apoptotic and anti-growth compound in diverse malignant tumors, including CRC. However, EUG's rapid metabolization, excretion, and side effects on normal cells at higher dosages are major limitations of its therapeutic potential. To address this problem, we developed a "smart" enzyme-responsive nanoparticle (eNP) loaded with EUG that exposes tumors to a high level of the drug while keeping its concentration low among healthy cells. We demonstrated that EUG induces apoptosis in CRC cells irrespective of their grades in a dose- and time-dependent manner. EUG significantly decreases cancer cell migration, invasion, and the population of colon cancer stem cells, which are key players in tumor metastasis and drug resistance. The "smart" eNPs-EUG show a high affinity to cancer cells with rapid internalization with no affinity toward normal colon epithelial cells. NPs-EUG enhanced the therapeutic efficacy of EUG measured by a cell viability assay and showed no toxicity effect on normal cells. The development of eNPs-EUG is a promising strategy for innovative anti-metastatic therapeutics.

A pH-/Enzyme-Responsive Nanoparticle Selectively Targets Endosomal Toll-like Receptors to Potentiate Robust Cancer Vaccination.

Toll-like receptor (TLR) agonists are potent immune-stimulators that hold great potential in vaccine adjuvants as well as cancer immunotherapy. However, TLR agonists in free form are prone to be eliminated quickly by the circulatory system and cause systemic inflammation side effects. It remains a challenge to achieve precise release of TLR7/8 agonist in the native form at the receptor site in the endosomal compartments while keeping stable encapsulation and inactive in nontarget environment. Here, we report a pH-/enzyme-responsive TLR7/8 agonist-conjugated nanovaccine (TNV), which responds intelligently to the acidic environment and cathepsin B in the endosome, precisely releases TLR7/8 agonist to activate its receptor signaling at the endosomal membrane, stimulates DCs maturation, and provokes specific cellular immunity. In vivo experiments demonstrate outstanding prophylactic and therapeutic efficacy of TNV in mouse melanoma and colon cancer. The endosome-targeted responsive nanoparticle strategy provides a potential delivery toolbox of adjuvants to advance the development of tumor nanovaccines.

Enzyme-Responsive Nanoparticles and Coatings Made from Alginate/Peptide Ciprofloxacin Conjugates as Drug Release System.

Infection-controlled release of antibacterial agents is of great importance, particularly for the control of peri-implant infections in the postoperative phase. Polymers containing antibiotics bound via enzymatically cleavable linkers could provide access to drug release systems that could accomplish this. Dispersions of nanogels were prepared by ionotropic gelation of alginate with poly-l-lysine, which was conjugated with ciprofloxacin as model drug via a copper-free 1,3-dipolar cycloaddition (click reaction). The nanogels are stable in dispersion and form films which are stable in aqueous environments. However, both the nanogels and the layers are degraded in the presence of an enzyme and the ciprofloxacin is released. The efficacy of the released drug against Staphylococcus aureus is negatively affected by the residues of the linker. Both the acyl modification of the amine nitrogen in ciprofloxacin and the sterically very demanding linker group with three annellated rings could be responsible for this. However the basic feasibility of the principle for enzyme-triggered release of drugs was successfully demonstrated.

Targeted apoptosis of macrophages and osteoclasts in arthritic joints is effective against advanced inflammatory arthritis

Insufficient apoptosis of inflammatory macrophages and osteoclasts (OCs) in rheumatoid arthritis (RA) joints contributes toward the persistent progression of joint inflammation and destruction. Here, we deliver celastrol (CEL) to selectively induce apoptosis of OCs and macrophages in arthritic joints, with enzyme-responsive nanoparticles (termed PRNPs) composed of RGD modified nanoparticles (termed RNPs) covered with cleavable PEG chains. CEL-loaded PRNPs (CEL-PRNPs) dually target OCs and inflammatory macrophages derived from patients with RA via an RGD-αvβ3 integrin interaction after PEG cleavage by matrix metalloprotease 9, leading to increased apoptosis of these cells. In an adjuvant-induced arthritis rat model, PRNPs have an arthritic joint-specific distribution and CEL-PRNPs efficiently reduce the number of OCs and inflammatory macrophages within these joints. Additionally, rats with advanced arthritis go into inflammatory remission with bone erosion repair and negligible side effects after CEL-PRNPs treatment. These findings indicate potential for targeting chemotherapy-induced apoptosis in the treatment of advanced inflammatory arthritis.

Enzyme-Responsive Nanoparticles for Anti-tumor Drug Delivery.

The past few decades have seen great progress in the exploration of nanoparticles (NPs) as novel tools for cancer treatments and diagnosis. Practical and reliable application of nanoparticle-based technology in clinical transformation remains nevertheless an ongoing challenge. The design, preparation, and evaluation of various smart NPs with specific physicochemical responses in tumor-related physiological conditions have been of great interests in both academic and clinical research. Of particular, smart enzyme-responsive nanoparticles can predictively and selectively react with specific enzymes expressed in tumor tissues, leading to targeted delivery of anti-tumor drugs, reduced systemic toxicity, and improved therapeutic effect. In addition, NPs interact with internal enzymes usually under mild conditions (low temperature, aqueous media, neutral or close to neutral pH) with high efficiency. In this review, recent advances in the past 5 years in enzyme-responsive nanoparticles for anti-tumor drug delivery are summarized and discussed. The following contents are divided based on the different action sites of enzymes toward NPs, notably hydrophobic core, cleavable/uncleavable linker, hydrophilic crown, and targeting ligand. Enzyme-engaged destruction of any component of these delicate nanoparticle structures could result in either targeting drug delivery or controlled drug release.

Enhancement in Site-Specific Delivery of Carvacrol against Methicillin Resistant Staphylococcus aureus Induced Skin Infections Using Enzyme Responsive Nanoparticles: A Proof of Concept Study.

Methicillin resistant Staphylococcus aureus (MRSA) induced skin infections have become a challenging problem due to the escalating antibiotic resistance. Carvacrol (CAR) has been reported to be effective against MRSA. However, due to its characteristics, CAR exhibits low skin retention. In this study, CAR was formulated into site-specific nanoparticle (NPs) delivery system using poly(ε-caprolactone) (PCL), following incorporation into a hydrogel matrix to facilitate dermal delivery. The release study exhibited significantly higher release of CAR from PCL NPs in the presence of bacterial lipase, highlighting its potential for differential delivery. Moreover, encapsulation of CAR in PCL NPs resulted in a two-fold increase in its anti-MRSA activity. Dermatokinetic studies revealed that the NPs loaded hydrogel was able to enhance skin retention of CAR after 24 h (83.29 ± 3.15%), compared to free CAR-loaded hydrogel (0.85 ± 0.14%). Importantly, this novel approach exhibited effective antimicrobial activity in an ex-vivo skin infection model. Hence, these findings have proven the concept that the loading of CAR into a responsive NPs system can lead to sustained antimicrobial effect at the desired site, and may provide a novel effective approach for treatment of MRSA induced skin infections. However, further studies must be conducted to investigate in-vivo efficacy of the developed system in an appropriate infection model.

Enzyme responsiveness enhances the specificity and effectiveness of nanoparticles for the treatment of B16F10 melanoma

The clinical treatment of melanoma continues to present many challenges including poor prognosis because neither monotherapy nor combination therapies have shown maximal treatment efficacy. In this study, an enzyme-responsive nanoparticle was designed for tumor subtypes with the high expression of heparanase-1, since highly metastatic tumors such as melanoma generally express significant levels of heparanase-1. PTX-DOTAP@alloferon-1-heparin/protamine, an enzyme-responsive nanoparticle, has a particle size of 106.1 ± 1.113 nm and a ζ-potential of -45.1 ± 0.455 mV, which enables enrichment in the tumor site by passive targeting. Subsequently, heparanase-1, which is highly expressed in the extracellular matrix, rapidly recognizes and degrades heparin in the outer layer of the nanoparticle and releases encapsulated alloferon-1 by ion diffusion to activate inhibited NK cells in the tumor microenvironment. The size of the smart nanoparticle will eventually decrease to 59.30 ± 0.783 nm and the ζ-potential will reverse to 25.4 ± 0.257 mV, which is beneficial for deep penetration and tumor cell uptake (due to the high negative charge on the tumor cell surface) of PTX-DOTAP cores. Paclitaxel is released in the cytoplasm, and the tumor cells are arrested in the G2/M phase. The nanoparticle characterization experiment demonstrated that in vivo drug delivery could be completed. In subsequent cell and animal experiments, the experimental data demonstrated the efficient therapeutic effects of the nanoparticle. This study provides an excellent template nanoparticle for the treatment of highly metastatic tumors to enhance future prognosis.

Delivery of Immunotherapeutic Nanoparticles to Tumors via Enzyme-Directed Assembly.

Amphiphilic diblock copolymers are prepared by ring opening metathesis polymerization, with one block containing hydrophobic Toll-like receptor 7 (TLR7) agonists and one block containing hydrophilic peptides as substrates for matrix metalloproteinases (MMPs). A fluorescent label is incorporated into the polymer chains for in vivo imaging. Upon dialysis against aqueous solution, polymers form 15 nm spherical micelles. Subsequent exposure to MMP-9 elicits a morphological change to yield immunostimulatory microscale assemblies. The intravenous (IV) administration of the formulation to mice bearing 4T1 breast cancer tumors results in nanoparticle accumulation in tumors, reduction in primary tumor growth, and inhibition of lung metastases, as compared to saline-treated animals. Mice administered the parent immunotherapeutic small molecule (1V209) experience significantly increased plasma levels of proinflammatory cytokines IL-6, IP-10, and MCP-1 at 2 h following IV administration, whereas the nanomaterial shows no increase over saline-treated controls. These data suggest that covalently packaging low molecular weight immunotherapeutics at high weight percent loadings in enzyme-responsive nanoparticles maintains drug efficacy while decreasing immunotoxicity, providing a platform for cancer immunotherapeutic delivery.

PEGylated self-assembled enzyme-responsive nanoparticles for effective targeted therapy against lung tumors

BackgroundMatrix-metalloproteinases, which are overexpressed in many types of cancer, can be applied to improve the bioavailability of chemotherapeutic drugs and guide therapeutic targeting. Thus, we aimed to develop enzyme-responsive nanoparticles based on a functionalized copolymer (mPEG-Peptide-PCL), which was sensitive to matrix metalloproteinase, as smart drug vesicles for enhanced biological specificity and reduced side effects.ResultsThe rate of in vitro curcumin (Cur) release from Cur-P-NPs was not markedly accelerated in weakly acidic tumor microenvironment, indicating a stable intracellular concentration and a consistent therapeutic effect. Meanwhile, P-NPs and Cur-P-NPs displayed prominent biocompatibility, biostability, and inhibition efficiency in tumor cells. In addition, Cur-P-NPs showed higher fluorescence intensity than Cur-NPs in tumor cells, implying enhanced cell permeability and targeting ability. Moreover, the internalization and intracellular transport of Cur-P-NPs were mainly via macropinocytosis. Studies of pharmacodynamics and cellular uptake in vitro and biodistribution in vivo demonstrated that Cur-P-NPs had stronger target efficiency and therapeutic effect than Cur-DMSO and Cur-NPs in tumor tissue.ConclusionResults indicate that Cur-P-NPs can be employed for active targeted drug delivery in cancer treatment and other biomedical applications.

Abstract 4664: Enzymatically-responsive tumor-targeted mesoporous silica nanoparticle for identification of pancreatic cancer

Abstract Purpose: Pancreatic cancer remains an unsolved health issue, with its rapid progression and resistance to modern therapy leading to poor prognoses for most patients. The prevalence of metastasis in pancreatic cancer makes complete tumor location and removal rare, and those who do have resectable disease have only a 20% 5-year survival rate. In recent years, nanoparticles have been explored as targeted delivery agents for chemotherapeutic drugs and imaging dyes, however few have achieved clinical success due to overestimation of biological phenomena and reliance on passive targeting systems. In this work, an enzyme-responsive nanoparticle has been developed for increased active pancreatic tumor targeting and specific release at biologically-significant enzyme concentrations. Methods: Mesoporous silica nanoparticles were formed using a scaffold of hexadecyltrimethylammonium bromide (CTAB) at 80°C. The scaffold was removed to form wormhole-like pores using a series of dialysis procedures. Transmission electron microscopy (TEM) confirmed the 35 nm diameter and porous structure of the particles. The particles were then loaded with IR780 dye, and surfaces were functionalized with (3-aminopropyl) triethoxysilane (APTES). The loaded particles were then encapsulated with a combination of Type A and Type B gelatin, followed by a stabilizing polyvinylpyrrolidone (PVP) layer. Dynamic light scattering (DLS) and zeta potential were used to confirm coating. Coated particle samples were exposed to collagenase type IV (MMP-9) enzyme to test encapsulation efficiency and enzyme sensitivity. Enzyme-treated and intact samples were transferred to tissue phantoms, and Multispectral optoacoustic tomography (MSOT) was used for comparative analysis. Results: TEM confirmed the formation of stable 35 nm silica nanoparticles with a wormhole-like pore structure. Zeta potential decrease from 55 mV to 5 mV and DLS particle diameter increase from 35 nm to 334.5 nm indicated binding of gelatin and PVP to particle surfaces. MSOT imaging showed 10 X increased signals from untreated nanoparticles as compared to enzyme-treated nanoparticles, indicating that dye molecules remained inside the pores of coated particles and were released when exposed to MMP-9. Conclusion: Gelatin/PVP-coated mesoporous silica nanoparticles encapsulated dye and demonstrated MMP-9 activated dye release at biologically-relevant enzyme concentrations. Citation Format: Kylie Nairon, Abhilash Samykutty, Molly W. McNally, Girish Mishra, William E. Grizzle, Lacey R. McNally. Enzymatically-responsive tumor-targeted mesoporous silica nanoparticle for identification of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4664.

Advancements in nanotechnology for drug delivery

Background: Nanoparticles (NPs) have emerged as a transformative technology in drug delivery, offering advancements in precision medicine, especially in managing chronic diseases and gastrointestinal (GIT) disorders. Due to their unique properties and the ability to be engineered at the nanoscale, NPs provide enhanced targeting, controlled release, and reduced side effects compared to traditional drug delivery systems. Aim: This review aims to summarize recent innovations in nanoparticle technology and their applications in drug delivery systems, with a focus on gastrointestinal diseases. Methods: The review synthesizes current literature on NP technologies and their applications in treating GIT disorders. It covers a range of nanocarriers, including metal and polymeric NPs, liposomes, hydrogels, and lipid nanoparticles. The review evaluates their effectiveness, challenges, and advancements in GIT drug delivery. Results: Recent advancements in NP technology have demonstrated significant potential in improving drug delivery to the GIT. Innovations include pH-sensitive NPs, enzyme-responsive NPs, and targeted formulations for diseases such as inflammatory bowel disease (IBD) and colorectal cancer. Lipid nanoparticles, including solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs), have shown promise in enhancing stability, targeting, and drug release profiles.

Enzyme-Responsive Nanoparticles for the Treatment of Disease.

Nanomedicine for cancer therapy seeks to treat malignancies through the selective accumulation of therapeutics in diseased tissue. Nanoparticles offer the convenience of high drug loading capacities and can be readily decorated with targeting moieties, drugs, and/or diagnostics. Our lab has pioneered a new tissue targeting strategy where enhanced accumulation of nanomaterials occurs as a result of morphology changes to the material in response to overexpressed enzymes in diseased tissues. Herein, we describe the general strategy for the preparation of these enzyme-responsive nanoparticles (ER-NPs) for therapeutic applications.

Enzyme-responsive Nanoparticles for Anticancer Drug Delivery

Enzyme-responsive Nanoparticles for Anticancer Drug Delivery

Therapeutic Enzyme-Responsive Nanoparticles for Targeted Delivery and Accumulation in Tumors.

An enzyme-responsive, paclitaxel-loaded nanoparticle is described and assessed in vivo in a human fibrosarcoma murine xenograft. This work represents a proof-of-concept study demonstrating the utility of enzyme-responsive nanoscale drug carriers capable of targeted accumulation and retention in tumor tissue in response to overexpressed endogenous enzymes.

Enzyme-Directed Assembly of Nanoparticles in Tumors Monitored by in Vivo Whole Animal Imaging and ex Vivo Super-Resolution Fluorescence Imaging

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO/DMF to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micrometer-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super-resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

Biogenic Growth of Alloys and Core-Shell Nanostructures Using Urease as a Nanoreactor at Ambient Conditions

Biomineralization is an extremely efficient biologically guided process towards the advancement of nano-bio integrated materials. As a prime module of the natural world, enzymes are expected to play a major role in biogenic growth of inorganic nanostructures. Although there have been developments in designing enzyme-responsive nanoparticle systems or generation of inorganic nanostructures in an enzyme-stimulated environment, reports regarding action of enzymes as reducing agents themselves for the growth of inorganic nanoparticles still remains elusive. Here we present a mechanistic investigation towards the synthesis of metal and metallic alloy nanoparticles using a commonly investigated enzyme, Jack bean urease (JBU), as a reducing as well as stabilizing agent under physiological conditions. The catalytic functionality of urease was taken advantage of towards the development of metal-ZnO core-shell nanocomposites, making urease an ideal bionanoreactor for synthesizing higher order nanostructures such as alloys and core- shell under ambient conditions.