ABSTRACT
Advanced staged high-grade serous ovarian cancer (HGSOC) is the leading cause of gynecological cancer death in the developed world, with 5-year survival rates of only 25-30% due to late-stage diagnosis and the shortcomings of platinum-based therapies. A Phase I clinical trial of a combination of free cisplatin and poly(ADP-ribose) polymerase inhibitors (PARPis) showed therapeutic benefit for HGSOC. In this study, we address the challenge of resistance to platinum-based therapy by developing a targeted delivery approach. Novel electrostatic layer-by-layer (LbL) liposomal nanoparticles (NPs) with a terminal hyaluronic acid layer that facilitates CD44 receptor targeting are designed for selective targeting of HGSOC cells; the liposomes can be formulated to contain both cisplatin and the PARPi drug within the liposomal core and bilayer. The therapeutic effectiveness of LbL NP-encapsulated cisplatin and PARPi alone and in combination was compared with the corresponding free drugs in luciferase and CD44-expressing OVCAR8 orthotopic xenografts in female nude mice. The NPs exhibited prolonged blood circulation half-life, mechanistic staged drug release and targeted codelivery of the therapeutic agents to HGSOC cells. Moreover, compared to the free drugs, the NPs resulted in significantly reduced tumor metastasis, extended survival, and moderated systemic toxicity.
ABSTRACT
DNA damaging chemotherapy is a cornerstone of current front-line treatments for advanced ovarian cancer (OC). Despite the fact that a majority of these patients initially respond to therapy, most will relapse with chemo-resistant disease; therefore, adjuvant treatments that synergize with DNA-damaging chemotherapy could improve treatment outcomes and survival in patients with this deadly disease. Here, we report the development of a nanoscale peptide-nucleic acid complex that facilitates tumor-specific RNA interference therapy to chemosensitize advanced ovarian tumors to frontline platinum/taxane therapy. We found that the nanoplex-mediated silencing of the protein kinase, MK2, profoundly sensitized mouse models of high-grade serous OC to cytotoxic chemotherapy by blocking p38/MK2-dependent cell cycle checkpoint maintenance. Combined RNAi therapy improved overall survival by 37% compared with platinum/taxane chemotherapy alone and decreased metastatic spread to the lungs without observable toxic side effects. These findings suggest (a) that peptide nanoplexes can serve as safe and effective delivery vectors for siRNA and (b) that combined inhibition of MK2 could improve treatment outcomes in patients currently receiving frontline chemotherapy for advanced OC.
ABSTRACT
Novel painkillers are urgently needed. The activation of opioid receptors in peripheral inflamed tissue can reduce pain without central adverse effects such as sedation, apnoea, or addiction. Here, we use an unprecedented strategy and report the synthesis and analgesic efficacy of the standard opioid morphine covalently attached to hyperbranched polyglycerol (PG-M) by a cleavable linker. With its high-molecular weight and hydrophilicity, this conjugate is designed to selectively release morphine in injured tissue and to prevent blood-brain barrier permeation. In contrast to conventional morphine, intravenous PG-M exclusively activated peripheral opioid receptors to produce analgesia in inflamed rat paws without major side effects such as sedation or constipation. Concentrations of morphine in the brain, blood, paw tissue, and in vitro confirmed the selective release of morphine in the inflamed milieu. Thus, PG-M may serve as prototype of a peripherally restricted opioid formulation designed to forego central and intestinal side effects.
Subject(s)
Analgesia/methods , Analgesics/pharmacology , Analgesics/pharmacokinetics , Glycerol/pharmacology , Glycerol/pharmacokinetics , Morphine/pharmacology , Morphine/pharmacokinetics , Polymers/pharmacology , Polymers/pharmacokinetics , Analgesics/chemistry , Animal Structures/chemistry , Animals , Glycerol/chemistry , Hydrophobic and Hydrophilic Interactions , Molecular Weight , Morphine/chemistry , Polymers/chemistry , RatsABSTRACT
All-trans retinoic acid (ATRA), a derivative of vitamin A, is a common component in cosmetics and commercial acne creams as well as being a first-line chemotherapeutic agent. Today, formulations for the topical application of ATRA rely on creams and emulsions to incorporate the highly hydrophobic ATRA drug. These strategies, when applied to the skin, deliver ATRA as a single bolus, which is immediately taken up into the skin and contributes to many of the known adverse side effects of ATRA treatment, including skin irritation and hair loss. Herein we present a new concept in topical delivery of retinoids by covalently bonding the drug through a hydrolytically degradable ester linkage to a common hydrophilic polymer, polyvinyl alcohol (PVA), creating an amphiphilic nanomaterial that is water-soluble. This PVA bound ATRA can then act as a pro-drug and accumulate within the skin to allow for the sustained controlled delivery of active ATRA. This approach was demonstrated to release active ATRA out to 10days in vitro while significantly enhancing dermal accumulation of the ATRA in explant pig skin. In vivo we demonstrate that the pro-drug formulation reduces application site inflammation compared to free ATRA and retains the drug at the application site at measurable quantities for up to six days.
Subject(s)
Nanostructures/administration & dosage , Polyvinyl Alcohol/administration & dosage , Prodrugs/administration & dosage , Tretinoin/administration & dosage , Administration, Cutaneous , Animals , Cell Survival/drug effects , Delayed-Action Preparations/administration & dosage , Delayed-Action Preparations/chemistry , Delayed-Action Preparations/pharmacokinetics , Drug Liberation , Female , Mice , NIH 3T3 Cells , Nanostructures/chemistry , Polyvinyl Alcohol/chemistry , Polyvinyl Alcohol/pharmacokinetics , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Skin/metabolism , Swine , Tretinoin/chemistry , Tretinoin/pharmacokineticsABSTRACT
A ligand decorated, synthetic polypeptide block copolymer platform with environment-responsive capabilities was designed. We evaluated the potential of this system to function as a polymersome for targeted-delivery of a systemic chemotherapy to tumors. Our system employed click chemistry to provide a pH-responsive polypeptide block that drives nanoparticle assembly, and a ligand (folic acid) conjugated PEG block that targets folate-receptor over-expressing cancer cells. These nanocarriers were found to encapsulate a high loading of conventional chemotherapeutics (e.g. doxorubicin at physiological pH) and release the active therapeutic at lysosomal pH upon cellular uptake. The presence of folic acid on the nanoparticle surface facilitated their active accumulation in folate-receptor-overexpressing cancer cells (KB), compared to untargeted carriers. Folate-targeted nanoparticles loaded with doxorubicin also showed enhanced tumor accumulation in folate-receptor positive KB xenografts, resulting in the suppression of tumor growth in an in vivo hind flank xenograft mouse model.
Subject(s)
Antibiotics, Antineoplastic/administration & dosage , Doxorubicin/administration & dosage , Drug Delivery Systems , Nanoparticles , Peptides , Animals , Cell Line, Tumor , Drug Carriers , Folic Acid , Humans , Mice , PolymersABSTRACT
Layer-by-layer (LbL) self-assembly is a versatile technique from which multicomponent and stimuli-responsive nanoscale drug carriers can be constructed. Despite the benefits of LbL assembly, the conventional synthetic approach for fabricating LbL nanoparticles requires numerous purification steps that limit scale, yield, efficiency, and potential for clinical translation. In this report, we describe a generalizable method for increasing throughput with LbL assembly by using highly scalable, closed-loop diafiltration to manage intermediate purification steps. This method facilitates highly controlled fabrication of diverse nanoscale LbL formulations smaller than 150 nm composed from solid-polymer, mesoporous silica, and liposomal vesicles. The technique allows for the deposition of a broad range of polyelectrolytes that included native polysaccharides, linear polypeptides, and synthetic polymers. We also explore the cytotoxicity, shelf life and long-term storage of LbL nanoparticles produced using this approach. We find that LbL coated systems can be reliably and rapidly produced: specifically, LbL-modified liposomes could be lyophilized, stored at room temperature, and reconstituted without compromising drug encapsulation or particle stability, thereby facilitating large scale applications. Overall, this report describes an accessible approach that significantly improves the throughput of nanoscale LbL drug-carriers that show low toxicity and are amenable to clinically relevant storage conditions.
ABSTRACT
The efficient transport of biological therapeutic materials to target tissues within the body is critical to their efficacy. In cartilage tissue, the lack of blood vessels prevents the entry of systemically administered drugs at therapeutic levels. Within the articulating joint complex, the dense and highly charged extracellular matrix (ECM) hinders the transport of locally administered therapeutic molecules. Consequently, cartilage injury is difficult to treat and frequently results in debilitating osteoarthritis. Here we show a generalizable approach in which the electrostatic assembly of synthetic polypeptides and a protein, insulin-like growth factor-1 (IGF-1), can be used as an early interventional therapy to treat injury to the cartilage. We demonstrated that poly(glutamic acid) and poly(arginine) associated with the IGF-1 via electrostatic interactions, forming a net charged nanoscale polyelectrolyte complex (nanoplex). We observed that the nanoplex diffused into cartilage plugs in vitro and stimulated ECM production. In vivo, we monitored the transport, retention and therapeutic efficacy of the nanoplex in an established rat model of cartilage injury. A single therapeutic dose, when administered within 48 hours of the injury, conferred protection against cartilage degradation and controlled interleukin-1 (IL-1) mediated inflammation. IGF-1 contained in the nanoplex was detected in the joint space for up to 4 weeks following administration and retained bioactivity. The results indicate the potential of this approach as an early intervention therapy following joint injury to delay or even entirely prevent the onset of osteoarthritis.
ABSTRACT
Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell's ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 10(5) -10(10) doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell's ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4-12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug's single-cell potency and can be used for any fluorescent or fluorescently labeled drug, including nanoparticles or antibody-drug conjugates.
Subject(s)
Doxorubicin/pharmacology , Cell Division/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Colony-Forming Units Assay/methods , Coloring Agents/pharmacology , Flow Cytometry/methods , Fluorescent Dyes/pharmacology , HT29 Cells , Humans , Nanoparticles/administration & dosage , Verapamil/pharmacologyABSTRACT
Traumatic wounds and congenital defects that require large-scale bone tissue repair have few successful clinical therapies, particularly for craniomaxillofacial defects. Although bioactive materials have demonstrated alternative approaches to tissue repair, an optimized materials system for reproducible, safe, and targeted repair remains elusive. We hypothesized that controlled, rapid bone formation in large, critical-size defects could be induced by simultaneously delivering multiple biological growth factors to the site of the wound. Here, we report an approach for bone repair using a polyelectrolye multilayer coating carrying as little as 200 ng of bone morphogenetic protein-2 and platelet-derived growth factor-BB that were eluted over readily adapted time scales to induce rapid bone repair. Based on electrostatic interactions between the polymer multilayers and growth factors alone, we sustained mitogenic and osteogenic signals with these growth factors in an easily tunable and controlled manner to direct endogenous cell function. To prove the role of this adaptive release system, we applied the polyelectrolyte coating on a well-studied biodegradable poly(lactic-co-glycolic acid) support membrane. The released growth factors directed cellular processes to induce bone repair in a critical-size rat calvaria model. The released growth factors promoted local bone formation that bridged a critical-size defect in the calvaria as early as 2 wk after implantation. Mature, mechanically competent bone regenerated the native calvaria form. Such an approach could be clinically useful and has significant benefits as a synthetic, off-the-shelf, cell-free option for bone tissue repair and restoration.
Subject(s)
Bone Morphogenetic Protein 2/pharmacology , Bone Regeneration/drug effects , Proto-Oncogene Proteins c-sis/pharmacology , Regenerative Medicine/methods , Skull/drug effects , Wound Healing/drug effects , Alendronate/pharmacology , Angiogenesis Inducing Agents/pharmacology , Animals , Becaplermin , Biocompatible Materials/pharmacology , Bone Density Conservation Agents/pharmacology , Bone and Bones/drug effects , Disease Models, Animal , Lactic Acid/pharmacology , Male , Membranes, Artificial , Polyglycolic Acid/pharmacology , Polylactic Acid-Polyglycolic Acid Copolymer , Rats , Rats, Sprague-Dawley , Skull/injuriesABSTRACT
Herein we report the potential of click chemistry-modified polypeptide-based block copolymers for the facile fabrication of pH-sensitive nanoscale drug delivery systems. PEG-polypeptide copolymers with pendant amine chains were synthesized by combining N-carboxyanhydride-based ring-opening polymerization with post-functionalization using azide-alkyne cycloaddition. The synthesized block copolymers contain a polypeptide block with amine-functional side groups and were found to self-assemble into stable polymersomes and disassemble in a pH-responsive manner under a range of biologically relevant conditions. The self-assembly of these block copolymers yields nanometer-scale vesicular structures that are able to encapsulate hydrophilic cytotoxic agents like doxorubicin at physiological pH but that fall apart spontaneously at endosomal pH levels after cellular uptake. When drug-encapsulated copolymer assemblies were delivered systemically, significant levels of tumor accumulation were achieved, with efficacy against the triple-negative breast cancer cell line, MDA-MB-468, and suppression of tumor growth in an in vivo mouse model.
Subject(s)
Drug Carriers/chemistry , Endosomes/chemistry , Nanoparticles/chemistry , Peptides/chemistry , Polyethylene Glycols/chemistry , Polymers/chemistry , Animals , Cell Line, Tumor , Doxorubicin/chemistry , Drug Delivery Systems/methods , Humans , Hydrogen-Ion Concentration , Mice , Mice, Inbred BALB C , Polymerization , SolubilityABSTRACT
Translation of micelles from the laboratory to the clinic is limited by a poor understanding of their in vivo fate following administration. In this paper, we establish a robust approach to real-time monitoring of the in vivo stability of micelles using Förster Resonance Energy Transfer (FRET). This characterization method allows for exquisite insight into the fate of micellar constituents, affording the capabilities to rapidly and efficiently evaluate a library of synthetically derived micellar systems as new therapeutic platforms in vivo. FRET-enabled biological characterization further holds potential to tailor material systems being uniquely investigated across the delivery community towards the next generation of stable therapeutics for disease management.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Micelles , Polymers/chemistry , Animals , Female , Fluorescent Dyes/metabolism , Light , Mice , Mice, Inbred BALB C , Mice, Nude , Scattering, Radiation , Tissue DistributionABSTRACT
Current treatment options for debilitating bone diseases such as osteosarcoma, osteoporosis, and bone metastatic cancer are suboptimal and have low efficacy. New treatment options for these pathologies require targeted therapy that maximizes exposure to the diseased tissue and minimizes off-target side effects. This work investigates an approach for generating functional and targeted drug carriers specifically for treating primary osteosarcoma, a disease in which recurrence is common and the cure rate has remained around 20%. This approach utilizes the modularity of Layer-by-Layer (LbL) assembly to generate tissue-specific drug carriers for systemic administration. This is accomplished via surface modification of drug-loaded nanoparticles with an aqueous polyelectrolyte, poly(acrylic acid) (PAA), side-chain functionalized with alendronate, a potent clinically used bisphosphonate. Nanoparticles coated with PAA-alendronate are observed to bind and internalize rapidly in human osteosarcoma 143B cells. Encapsulation of doxorubicin, a front-line chemotherapeutic, in an LbL-targeted liposome demonstrates potent toxicity in vitro. Active targeting of 143B xenografts in NCR nude mice with the LbL-targeted doxorubicin liposomes promotes enhanced, prolonged tumor accumulation and significantly improved efficacy. This report represents a tunable approach towards the synthesis of drug carriers, in which LbL enables surface modification of nanoparticles for tissue-specific targeting and treatment.
Subject(s)
Drug Carriers/chemistry , Nanoparticles/chemistry , Acrylic Resins/chemistry , Alendronate/administration & dosage , Alendronate/chemistry , Animals , Bone Density Conservation Agents/administration & dosage , Bone Density Conservation Agents/chemistry , Bone Neoplasms/diagnostic imaging , Bone Neoplasms/drug therapy , Bone Neoplasms/pathology , Cell Line, Tumor , Doxorubicin/administration & dosage , Doxorubicin/analogs & derivatives , Doxorubicin/chemistry , Half-Life , Humans , Liposomes/chemistry , Liposomes/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Nanoparticles/metabolism , Osteosarcoma/diagnostic imaging , Osteosarcoma/drug therapy , Osteosarcoma/pathology , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/chemistry , Radiography , Transplantation, HeterologousABSTRACT
The functional success of a biomedical implant critically depends on its stable bonding with the host tissue. Aseptic implant loosening accounts for more than half of all joint replacement failures. Various materials, including metals and plastic, confer mechanical integrity to the device, but often these materials are not suitable for direct integration with the host tissue, which leads to implant loosening and patient morbidity. We describe a self-assembled, osteogenic, polymer-based conformal coating that promotes stable mechanical fixation of an implant in a surrogate rodent model. A single modular, polymer-based multilayered coating was deposited using a water-based layer-by-layer approach, by which each element was introduced on the surface in nanoscale layers. Osteoconductive hydroxyapatite (HAP) and osteoinductive bone morphogenetic protein-2 (BMP-2) contained within the nanostructured coating acted synergistically to induce osteoblastic differentiation of endogenous progenitor cells within the bone marrow, without indications of a foreign body response. The tuned release of BMP-2, controlled by a hydrolytically degradable poly(ß-amino ester), was essential for tissue regeneration, and in the presence of HAP, the modular coating encouraged the direct deposition of highly cohesive trabecular bone on the implant surface. In vivo, the bone-implant interfacial tensile strength was significantly higher than standard bioactive bone cement, did not fracture at the interface, and had long-term stability. Collectively, these results suggest that the multilayered coating system promotes biological fixation of orthopedic and dental implants to improve surgical outcomes by preventing loosening and premature failure.
Subject(s)
Coated Materials, Biocompatible/chemistry , Animals , Bone Morphogenetic Protein 2/chemistry , Bone Morphogenetic Protein 2/pharmacology , Bone Regeneration/drug effects , Coated Materials, Biocompatible/pharmacology , Durapatite/chemistry , Durapatite/pharmacology , Male , Osteogenesis/drug effects , Polymers/chemistry , Polymers/pharmacology , Prostheses and Implants , Rats , Rats, Sprague-DawleyABSTRACT
The use of polymeric nanocarriers to transport active compounds like small-molecular drugs, peptides, or genes found an increased attention throughout the different fields of natural sciences. Not only that these nanocarriers enhance the properties of already existing drugs in terms of solubility, bioavailability, and prolonged circulation times, furthermore they can be tailor-made in such a manner that they selectively release their cargo at the desired site of action. For the triggered release, these so-called smart drug delivery systems are designed to react on certain stimuli like pH, temperature, redox potential, enzymes, light, and ultrasound. Some of these stimuli are naturally occurring in vivo, for example the difference in pH in different cellular compartments while others are caused by the disease, which is to be treated, like differences in pH and temperature in some tumor tissues. Other external applied stimuli, like light and ultrasound, allow the temporal and spatial control of the release, since they are not triggered by any biological event. This review gives a brief overview about some types of stimuli-responsive nanocarriers with the main focus on organic polymer-based systems. Furthermore, the different stimuli and the design of corresponding responsive nanocarriers will be discussed with the help of selected examples from the literature.
Subject(s)
Drug Carriers/administration & dosage , Drug Carriers/chemistry , Drug Delivery Systems/methods , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Polymers/administration & dosage , Polymers/chemistry , Animals , Biological Transport , HumansABSTRACT
Among the various polymeric architectures, dendritic polymers have received a substantial scientific focus for their highly branched, multifunctional, and well-defined structures. Dendritic scaffolds have found many applications for designing nanoscale drug/gene delivery carriers and constructing diagnostic and biosensor devices, and protein-resistant surfaces. A significant number of research groups across Europe share the common objective, yet in conspicuously individual ways, of utilizing such polymers for devising innovative biomedical tools and techniques. This review describes the European effort of finding the application of dendritic polymers as advanced generation therapeutics within the purview of nanomedicine.
Subject(s)
Dendrimers/therapeutic use , Drug Delivery Systems , Gene Transfer Techniques , Nanoparticles/therapeutic use , Animals , Anti-Inflammatory Agents , HumansABSTRACT
Two photo-responsive core/shell nanoparticles based on hyperbranched polyglycerol (hPG) are synthesized for controlled release of DNA. The shell is composed either of bis-(3-aminopropyl)methylamine (AMPA) or pentaethylenehexamine (PEHA) derivatives and is attached to the hPG core with a photo-responsive o-nitrobenzyl linker. Ethidium bromide displacement assay, gel electrophoresis, DLS, and ζ-potential measurements are performed with these nanoparticles. Photo-responsive changes within the carrier scaffold are investigated by irradiating the polymer solution with 350 nm monochromatic light. Fully covered APMA-shelled carriers are found to complex the DNA at an N/P ratio of 10 with an average size ranging from 54 to 78 nm depending on the degree of functionalization of the core.
Subject(s)
DNA/chemistry , Gene Transfer Techniques , Glycerol/chemistry , Methylamines/chemistry , Polyamines/chemistry , Polymers/chemistry , DNA/administration & dosage , Delayed-Action Preparations , Electrophoretic Mobility Shift Assay , Endocytosis , HeLa Cells , Humans , Nanoparticles , Photoelectron Spectroscopy , Ultraviolet RaysABSTRACT
Stimuli-responsive polymer architectures are molecular systems which evolve with an external signal. The observed changes are mainly decomposition, isomerization, polymerization, activation, supramolecular aggregation, and structural modifications of these molecules. The external stimuli, which can be combined in order to provoke these molecular changes, are numerous. In this review, we have chosen to present an overview on different mechanisms to impart responsiveness to dendritic polymers, with the particular aim of delivery and release of bioactive molecules.
Subject(s)
Dendrimers/chemistry , Drug Carriers/chemistry , Hydrogen-Ion Concentration , Nanotechnology/methods , Oxidation-ReductionABSTRACT
Here we present the efficiency and versatility of newly developed core-multishell nanoparticles (CMS NPs), to encapsulate and transport the antitumor drugs doxorubicin hydrochloride (Dox), methotrexate (Mtx) and sodium ibandronate (Ibn) as well as dye molecules, i.e., a tetrasulfonated indotricarbocyanine (ITCC) and nile red. Structurally, the CMS NPs are composed of hyperbranched poly(ethylene imine) core functionalized by alkyl diacids connected to monomethyl poly(ethylene glycol). In order to evaluate their transport in aqueous media in vitro, we have used and compared SEC, UV, ITC, and NMR techniques. We observed that the CMS NPs were able to spontaneously encapsulate and transport Dox, Mtx and nile red in both organic and aqueous media as determined by SEC and UV-VIS spectroscopy. For the VIS transparent Ibn Isothermal Titration Calorimetric (ITC) experiments show an exothermic interaction with the CMS NPs. The enthalpic stabilization (DeltaH) upon encapsulation was in the order of approximately 7 kcals/mol which indicates stable interaction between Ibn and nanoparticles. A T(1) inversion recovery NMR experiment was carried out for 31P and 1H nuclei of Ibn and an increment of spin-lattice relaxation time for respective nuclei was observed upon encapsulation. CMS NPs were also found to encapsulate ITCC dye with stoichiometry of 6-8 molecules/nanocarrier. For in vivo imaging studies the dye loaded CMS NPs were injected to F9 teratocarcinoma bearing mice and a strong contrast was observed in the tumor tissues compared to free dye after 6 h of administration.