ABSTRACT
Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(ß-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigms via monocyte programming.
Subject(s)
Monocytes , Nanoparticles , RNA, Messenger , Monocytes/metabolism , Nanoparticles/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Animals , Mice , Humans , Polyelectrolytes/chemistry , Macrophages/metabolism , Polyamines/chemistry , Particle Size , Cell Differentiation , Gene Transfer Techniques , Dendritic Cells/metabolism , Static Electricity , PolymersABSTRACT
Aging negatively impacts skin health, notably through the senescent cell phenotype, which reduces collagen production and leads to thinner, more fragile skin prone to injuries and chronic wounds. We designed a drug delivery system that addresses these age-related issues using a hybrid hydrogel-nanoparticle system that utilizes a poly(δ-valerolactone-co-lactide)-b-poly(ethylene-glycol)-b-poly(δ-valerolactone-co-lactide) (PVLA-PEG-PVLA) hydrogel. This hydrogel allows for the local, extended release of therapeutics targeting both proliferating and senescent cells. The PVLA-PEG-PVLA hydrogel entrapped valsartan, and metformin-loaded liposomes functionalized with a fibronectin-mimetic peptide, PR_b. Metformin acts as a senomorphic, reversing aspects of cellular senescence, and valsartan, an angiotensin receptor blocker, promotes collagen production. This combination treatment partially reversed the senescent phenotype and improved collagen production in senescent dermal fibroblasts from both young and old adults. Our codelivery hydrogel-nanoparticle system offers a promising treatment for improving age-related dermal pathologies.
ABSTRACT
Lipid nanoparticles (LNP) have emerged as pivotal delivery vehicles for RNA therapeutics. Previous research and development usually assumed that LNPs are homogeneous in population, loading density, and composition. Such perspectives are difficult to examine due to the lack of suitable tools to characterize these physicochemical properties at the single-nanoparticle level. Here, we report an integrated spectroscopy-chromatography approach as a generalizable strategy to dissect the complexities of multicomponent LNP assembly. Our platform couples cylindrical illumination confocal spectroscopy (CICS) with single-nanoparticle free solution hydrodynamic separation (SN-FSHS) to simultaneously profile population identity, hydrodynamic size, RNA loading levels, and distributions of helper lipid and PEGylated lipid of LNPs at the single-particle level and in a high-throughput manner. Using a benchmark siRNA LNP formulation, we demonstrate the capability of this platform by distinguishing seven distinct LNP populations, quantitatively characterizing size distribution and RNA loading level in wide ranges, and more importantly, resolving composition-size correlations. This SN-FSHS-CICS analysis provides critical insights into a substantial degree of heterogeneity in the packing density of RNA in LNPs and size-dependent loading-size correlations, explained by kinetics-driven assembly mechanisms of RNA LNPs.
Subject(s)
Lipids , Nanoparticles , Particle Size , Nanoparticles/chemistry , Lipids/chemistry , RNA/chemistry , Chromatography/methods , RNA, Small Interfering/chemistry , Spectrum Analysis/methods , LiposomesABSTRACT
With its lack of commonly targeted receptors, triple negative breast cancer (TNBC) is aggressive and difficult to treat. To address this problem, nanotubes self-assembled from single stranded DNA (ssDNA)-amphiphiles were used as a delivery vehicle for doxorubicin (DOX) to target TNBC cells. Since DOX and other standard of care treatments such as radiation have been documented to induce senescence, the ability of the nanotubes to deliver the senolytic ABT-263 was also investigated. The ssDNA-amphiphiles were synthesized from a 10 nucleotide sequence attached to a dialkyl, (C16)2, tail via a C12 alkyl spacer, and have been previously shown to self-assemble into hollow nanotubes and spherical micelles. Here, we demontrate that these ssDNA spherical micelles could transition into long nanotubes in the presence of excess tails. The nanotubes could then be shortened via probe sonication. The ssDNA nanotubes internalized into three different TNBC cell lines: Sum159, MDA-MB-231, and BT549, with minimal internalization in healthy Hs578Bst cells, suggesting an inherent targeting ability. Inhibition of different internalization mechanisms showed that the nanotubes internalized in the TNBC cells primarily through macropinocytosis and scavenger receptor-mediated endocytosis, both of which are upregulated pathways in TNBC. DOX was intercalated into the ssDNA nanotubes and delivered to TNBC cells. Compared to free DOX, DOX-intercalated nanotubes proved equally cytotoxic to TNBC cells. In order to demonstrate the potential for delivery of different therapeutics, ABT-263 was incorporated into the hydrophobic bilayer wall of the nanotubes and was delivered to a DOX-induced in vitro model of senescence. The ABT-263 encapsulating nanotubes demonstrated cytotoxicity to senescent TNBC cells as well as sensitization to further DOX treatment. Thus, our ssDNA nanotubes are a promising delivery vehicle that could be used for targeted delivery of therapeutics to TNBC cells.
Subject(s)
Nanotubes , Triple Negative Breast Neoplasms , Humans , Micelles , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/metabolism , Senotherapeutics , DNA, Single-Stranded , Doxorubicin/pharmacology , Cell Line, Tumor , Nanotubes/chemistryABSTRACT
Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics. It has been challenging to assess mRNA packaging characteristics in LNPs, including payload distribution and capacity, which are critical to understanding structure-property-function relationships for further carrier development. Here, we report a method based on the multi-laser cylindrical illumination confocal spectroscopy (CICS) technique to examine mRNA and lipid contents in LNP formulations at the single-nanoparticle level. By differentiating unencapsulated mRNAs, empty LNPs and mRNA-loaded LNPs via coincidence analysis of fluorescent tags on different LNP components, and quantitatively resolving single-mRNA fluorescence, we reveal that a commonly referenced benchmark formulation using DLin-MC3 as the ionizable lipid contains mostly 2 mRNAs per loaded LNP with a presence of 40%-80% empty LNPs depending on the assembly conditions. Systematic analysis of different formulations with control variables reveals a kinetically controlled assembly mechanism that governs the payload distribution and capacity in LNPs. These results form the foundation for a holistic understanding of the molecular assembly of mRNA LNPs.
Subject(s)
Lipids , Nanoparticles , Lipids/chemistry , Liposomes , Nanoparticles/chemistry , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Small Interfering/geneticsABSTRACT
In this work, we demonstrate the formation of supramolecular architectures from the assembly of single-tail single stranded DNA (ssDNA)-amphiphiles. Short ssDNA sequences of 10 nucleotides that were either unstructured or formed G-quadruplex secondary structures were conjugated to a single 4-(hexadecyloxy)benzamide tail, either directly or through a polycarbon (C12) spacer. Conjugation of the ssDNA to the tail did not interfere with the G-quadruplex secondary structure of the ssDNA sequence. The ssDNA-amphiphiles self-assembled into ellipsoidal micelles, vesicles, nanotapes, and nanotubes. These nanotubes appeared to be formed by the rolling up of nanotapes. The increase of the hydrophobic block of the ssDNA-amphiphiles through the addition of a C12 spacer led to an increase in wall thickness and nanotube diameter. The presence of π-π interactions, through the benzoic group, was verified via X-ray diffraction (XRD) and played a critical role in the formation of the different nanostructures. In contrast, ssDNA-amphiphiles with a single heptadecanoic acid tail self-assembled only into ellipsoidal micelles.
Subject(s)
G-Quadruplexes , Nanotubes , DNA, Single-Stranded , Micelles , Hydrophobic and Hydrophilic Interactions , Nanotubes/chemistryABSTRACT
A DNA-based artificial metalloenzyme (ArM) consisting of a copper(II) complex of 4,4'-dimethyl-2,2'-bipyridine (dmbipy-Cu) bound to double-stranded DNA (dsDNA) as short as 8 base pairs with only 2 contiguous central pairs (G for guanine and C for cytosine) catalyzes the highly enantioselective Diels-Alder reaction, Michael addition, and Friedel-Crafts alkylation in water. Molecular simulations indicate that these minimal sequences provide a single site where dmbipy-Cu is groove-bound and able to function as an enantioselective catalyst. Enantioselective preference inverts when d-DNA is replaced with l-DNA. When the DNA is conjugated to a hydrophobic tail, the obtained ArMs exhibit enantioselective performance in a methanol-water mixture superior to that of non-amphiphilic dsDNA, and dsDNA-amphiphiles with more complex Gâ¢C-rich sequences.
ABSTRACT
Effective treatment of glioblastoma remains a daunting challenge. One of the major hurdles in the development of therapeutics is their inability to cross the blood-brain tumor barrier (BBTB). Local delivery is an alternative approach that can still suffer from toxicity in the absence of target selectivity. Here, we show that nanotubes formed from self-assembly of ssDNA-amphiphiles are stable in serum and nucleases. After bilateral brain injections, nanotubes show preferential retention by tumors compared to normal brain and are taken up by glioblastoma cells through scavenger receptor binding and macropinocytosis. After intravenous injection, they cross the BBTB and internalize in glioblastoma cells. In a minimal residual disease model, local delivery of doxorubicin showed signs of toxicity in the spleen and liver. In contrast, delivery of doxorubicin by the nanotubes resulted in no systemic toxicity and enhanced mouse survival. Our results demonstrate that ssDNA nanotubes are a promising drug delivery vehicle to glioblastoma.
ABSTRACT
Despite potential for clinical efficacy, therapeutic delivery of microRNAs (miRNA) remains a major translational barrier. Here, we explore a strategy for miRNA delivery in the treatment of glioblastoma, the most common form of adult brain cancer, that involves complexation of miRNA with polyethylenimine (PEI) and encapsulation in targeted liposomes. miRNA 603 (miR-603) is a master regulatory miRNA that suppresses glioblastoma radiation resistance through down-regulation of insulin-like growth factor 1 (IGF1) signaling. miR-603 was complexed with PEI, a cationic polymer, and encapsulated into liposomes decorated with polyethylene glycol (PEG) and PR_b, a fibronectin-mimetic peptide that specifically targets the α5ß1 integrin that is overexpressed in glioblastomas. Cultured patient-derived glioblastoma cells internalized PR_b-functionalized liposomes but not the non-targeted liposomes. The integrin targeting and complexation of the miRNA with PEI were associated with a 22-fold increase in intracellular miR-603 levels, and corresponding decreases in IGF1 and IGF1 receptor (IGF1R) mRNA expression. Moreover, treatment of glioblastoma cells with the PR_b liposomes encapsulating miR-603/PEI sensitized the cells to ionizing radiation (IR), a standard of care treatment for glioblastomas. These results suggest that PR_b-functionalized PEGylated liposomes encapsulating miR-603/PEI complexes hold promise as a therapeutic platform for glioblastomas.
ABSTRACT
Despite decades of research, there are few targeted treatment options available for triple negative breast cancer (TNBC), leaving chemotherapy, and radiation treatment regimes with poor response and high toxicity. Herein aptamer-amphiphiles were synthesized which selectively bind to the mucin-1 (MUC1) glycoprotein that is overexpressed in TNBC cells. These amphiphiles have a fluorescent tail (1,8-naphthalimide or 4-nitro-1,8-naphthalimide) which enables self-assembly of the amphiphiles and allows for easy visualization without the requirement for further conjugation of a fluorophore. Interestingly, the length of the alkyl spacer (C4 or C12) between the aptamer and tail was shown to influence the morphology of the self-assembled structure, and thus its ability to internalize into the TNBC cells. While both the MUC1 aptamer-C4-napthalimide spherical micelles and the MUC1 aptamer-C12-napthalimide long cylindrical micelles showed internalization into MDA-MB-468 TNBC cells but not the noncancerous MCF-10A breast cells, the cylindrical micelles showed greatly enhanced internalization into the MDA-MB-468 cells. Similar patterns of enhanced binding and internalization were observed between the MUC1 aptamer-C12-napthalimide cylindrical micelles and SUM159 and MDA-MB-231 TNBC cells. The MUC1 aptamer cylindrical micelles were not toxic to the cells, and when used to deliver doxorubicin to the TNBC cells, were shown to be as cytotoxic as free doxorubicin. Moreover, a pharmacokinetic study in mice showed a prolonged systemic circulation time of the MUC1 aptamer cylindrical micelles. There was a 4.6-fold increase in the elimination half-life of the aptamer cylindrical micelles, and their clearance decreased 10-fold compared to the MUC1 aptamer spherical micelles. Thus, the MUC1 aptamer-C12-napthalimide nanofibers represent a promising vehicle that could be used for easy visualization and targeted delivery of therapeutic loads to TNBC cells.
ABSTRACT
Pancreatic cancer represents a life threatening disease with rising mortality. Although the synergistic combination of gemcitabine and albumin-bound paclitaxel has proven to enhance the median survival rates as compared to gemcitabine alone, their systemic and repeated co-administration has been associated with serious toxic side effects and poor patient compliance. For this purpose, we designed a thermosensitive and biodegradable hydrogel encapsulating targeted nanoparticles for the local and sustained delivery of gemcitabine (GEM) and paclitaxel (PTX) to pancreatic cancer. GEM and PTX were loaded into PR_b-functionalized liposomes targeting integrin α5ß1, which was shown to be overexpressed in pancreatic cancer. PR_b is a fibronectin-mimetic peptide that binds to α5ß1 with high affinity and specificity. The PR_b liposomes were encapsulated into a poly(δ-valerolactone-co-D,L-lactide)-b-poly(ethylene glycol)-b-poly(δ-valerolactone-co-D,L-lactide) (PVLA-PEG-PVLA) hydrogel and demonstrated sustained release of both drugs compared to PR_b-functionalized liposomes free in solution or free drugs in the hydrogel. Moreover, the hydrogel-nanoparticle system was proven to be very efficient towards killing monolayers of human pancreatic cancer cells (PANC-1), and showed a significant reduction in the growth pattern of PANC-1 tumor spheroids as compared to hydrogels encapsulating non-targeted liposomes with GEM/PTX or free drugs, after a one week treatment period. Our hybrid hydrogel-nanoparticle system is a promising platform for the local and sustained delivery of GEM/PTX to pancreatic cancer, with the goal of maximizing the therapeutic efficacy of this synergistic drug cocktail while potentially minimizing toxic side effects and eliminating the need for repeated co-administration.
Subject(s)
Nanoparticles , Pancreatic Neoplasms , Cell Line, Tumor , Deoxycytidine/analogs & derivatives , Drug Delivery Systems , Humans , Hydrogels/therapeutic use , Paclitaxel/therapeutic use , Pancreatic Neoplasms/drug therapy , Polyethylene Glycols/therapeutic use , GemcitabineABSTRACT
BACKGROUND: Recurrence after radiation therapy is nearly universal for glioblastomas, the most common form of adult brain cancer. The study aims to define clinically pertinent mechanisms underlying this recurrence. METHODS: microRNA (miRNA) profiling was performed using matched pre- and post-radiation treatment glioblastoma specimens from the same patients. All specimens harbored unmethylated O6-methylguanine-DNA methyltransferase promoters (umMGMT) and wild-type isocitrate dehydrogenase (wtIDH). The most altered miRNA, miR-603, was characterized. FINDINGS: While nearly all miRNAs remained unchanged after treatment, decreased levels of few, select miRNAs in the post-treatment specimens were observed, the most notable of which involved miR-603. Unbiased profiling of miR-603 targets revealed insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R). Ionizing radiation (IR) induced cellular export of miR-603 through extracellular vesicle (EV) release, thereby de-repressing IGF1 and IGF1R. This de-repression, in turn, promoted cancer stem-cell (CSC) state and acquired radiation resistance in glioblastomas. Export of miR-603 additionally de-repressed MGMT, a DNA repair protein responsible for detoxifying DNA alkylating agents, to promote cross-resistance to these agents. Ectopic miR-603 expression overwhelmed cellular capacity for miR-603 export and synergized with the tumoricidal effects of IR and DNA alkylating agents. INTERPRETATION: Profiling of matched pre- and post-treatment glioblastoma specimens revealed altered homeostasis of select miRNAs in response to radiation. Radiation-induced EV export of miR-603 simultaneously promoted the CSC state and up-regulated DNA repair to promote acquired resistance. These effects were abolished by exogenous miR-603 expression, suggesting potential for clinical translation. FUNDING: NIH 1R01NS097649-01, 9R44GM128223-02, 1R01CA240953-01, the Doris Duke Charitable Foundation Clinical Scientist Development Award, The Sontag Foundation Distinguished Scientist Award, the Kimmel Scholar Award, and BWF 1006774.01 (C.C.C).
Subject(s)
Brain Neoplasms/genetics , DNA Modification Methylases/genetics , DNA Repair Enzymes/genetics , Extracellular Vesicles/radiation effects , Glioblastoma/genetics , Insulin-Like Growth Factor I/genetics , MicroRNAs/genetics , Radiation Tolerance/genetics , Tumor Suppressor Proteins/genetics , Animals , Brain Neoplasms/mortality , Brain Neoplasms/pathology , Brain Neoplasms/radiotherapy , Cell Line, Tumor , Cell Survival/radiation effects , DNA Modification Methylases/metabolism , DNA Repair/genetics , DNA Repair/radiation effects , DNA Repair Enzymes/metabolism , Extracellular Vesicles/genetics , Extracellular Vesicles/metabolism , Gamma Rays , Gene Expression Regulation, Neoplastic , Glioblastoma/mortality , Glioblastoma/pathology , Glioblastoma/radiotherapy , Histones/genetics , Histones/metabolism , Humans , Insulin-Like Growth Factor I/metabolism , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Male , Mice , Mice, Nude , MicroRNAs/metabolism , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Neoplastic Stem Cells/radiation effects , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Signal Transduction , Survival Analysis , Tumor Suppressor Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
An aptamer-amphiphile was designed that binds to ß-lactoglobulin (ß-LG), a major allergen from cow's milk. For this work, a 23-nucleotide ssDNA aptamer ß-LG-23, capable of forming antiparallel G-quadruplexes was used, and its specificity and binding affinity of 22 ± 2 nM for ß-LG were evaluated via enzyme-linked apta-sorbent assay (ELASA). The ß-LG-23 aptamer was synthesized as an amphiphile by conjugating it to a C16 double tail via different spacers, and the effect of the spacers on the binding affinity and secondary structure of the aptamer was investigated. From all amphiphiles tested, direct conjugation of the aptamer to the tail gave the lowest binding affinity to ß-LG (37 ± 2 nM), while maintaining the antiparallel G-quadruplex secondary structure of the aptamer. As a proof of concept, the ß-LG-23 aptamer-amphiphile was used to decorate the interface of a liquid crystal (LC) and effectively detected 10 nM or 0.18 ppm of ß-LG with a 20 min equilibration time, thus demonstrating that it has the potential to be used for fast and label-free detection of ß-LG.
Subject(s)
Aptamers, Nucleotide/chemistry , DNA, Single-Stranded/chemistry , Drug Design , Lactoglobulins/analysis , Liquid Crystals/chemistry , Animals , Aptamers, Nucleotide/genetics , Cattle , DNA, Single-Stranded/genetics , Lactoglobulins/chemistry , Lactoglobulins/geneticsABSTRACT
Controlling the dimensions of DNA nanotubes is of great interest as they can be used in different applications ranging from functional elements in nanodevices to carriers for drug delivery. ssDNA-amphiphiles composed of a ssDNA headgroup, a hydrophobic dialkyl tail and a polycarbon spacer between the tail and the headgroup, self-assemble into hollow DNA nanotubes by forming bilayer nanotapes that transition from twisted nanotapes, to helical nanotapes, to nanotubes. The presence of the DNA nanotubes is verified via cryo-TEM and SAXS. We further explore the effect of the ssDNA secondary structure and tail length on the assembly of the ssDNA-amphiphiles. We demonstrate that the presence of intermolecular G-quadruplexes in the ssDNA sequence dictates the nanotube length. The nanotube diameter is controlled by the hydrophobic tail length, and coarse-grained molecular dynamics simulations are employed to elucidate the tail design impact on assembly.
Subject(s)
DNA, Single-Stranded/chemistry , G-Quadruplexes , Nanotubes/chemistry , Nanotubes/ultrastructureABSTRACT
In this work we hypothesized that the chemokine fractalkine can serve as a cancer molecular target. We engineered aptamer micelles functionalized with an outer poly(ethylene glycol) (PEG) corona, and investigated the extent and efficacy of using them as a targeting tool against fractalkine-expressing colon adenocarcinoma cells. In vitro cell binding results showed that aptamer micelles bound and internalized to fractalkine-expressing cancer cells with the majority of the micelles found free in the cytoplasm. Minimal surface binding was observed by healthy cells. Even though partial PEGylation did not prevent serum adsorption, micelles were highly resistant to endonuclease and exonuclease degradation. In vivo biodistribution studies and confocal studies demonstrated that even though both aptamer and control micelles showed tumor accumulation, only the aptamer micelles internalized into fractalkine-expressing cancer cells, thus demonstrating the potential of the approach and showing that fractalkine may serve as a specific target for nanoparticle delivery to cancer cells.
Subject(s)
Adenocarcinoma/drug therapy , Aptamers, Nucleotide/administration & dosage , Chemokine CX3CL1/metabolism , Colonic Neoplasms/drug therapy , Fibroblasts/drug effects , Micelles , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Aptamers, Nucleotide/chemistry , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Drug Carriers/chemistry , Drug Delivery Systems , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , In Vitro Techniques , Mice , Polyethylene Glycols/chemistry , Tumor Cells, CulturedABSTRACT
A comprehensive study is reported on the effect of salt concentration, polyelectrolyte block length, and polymer concentration on the morphology and structural properties of nanoaggregates self-assembled from BAB single-strand DNA (ssDNA) triblock polynucleotides in which A represents polyelectrolyte blocks and B represents hydrophobic neutral blocks. A morphological phase diagram above the gelation point is developed as a function of solvent ionic strength and polyelectrolyte block length utilizing an implicit solvent ionic strength method for dissipative particle dynamics simulations. As the solvent ionic strength increases, the self-assembled DNA network structures shrinks considerably, leading to a morphological transition from a micellar network to worm-like or hamburger-shape aggregates. This study provides insight into the network morphology and its changes by calculating the aggregation number, number of hydrophobic cores, and percentage of bridge chains in the network. The simulation results are corroborated through cryogenic transmission electron microscopy on the example of the self-assembly of ssDNA triblocks.
Subject(s)
DNA, Single-Stranded/chemistry , Polyelectrolytes/chemistry , Sodium Chloride/chemistry , Cryoelectron Microscopy , Hydrophobic and Hydrophilic Interactions , Micelles , Osmolar Concentration , Solvents/chemistryABSTRACT
Nanoparticles functionalized with cancer-targeting ligands have shown promise but are still limited by off-tumor binding to healthy tissues that express low levels of the molecular target. Targeting two cancer biomarkers using dual-targeted heteromultivalent nanoparticles presents a possible solution to this challenge by requiring overexpression of two separate ligands for localization. In order to guide experimental design, a kinetic model was built to explore how the affinity and valency of dual-ligand liposomes affect the binding and selectivity of delivery to cells with various receptor expression. α5ß1 and α6ß4 integrin expression levels were quantified on 20 different cell lines to identify appropriate model cells for in vitro investigation. Dual-targeting heteromultivalent liposomes covered with polyethylene glycol (PEG) were synthesized using the PR_b peptide that binds to the α5ß1 integrin and the AG86 peptide that binds to the α6ß4 integrin. PEGylated liposomes with varying ratios of the targeting peptides were delivered to cells with different integrin concentrations. Nanoparticle binding and internalization as well as integrin internalization as a function of time were evaluated to understand the effect of valency and avidity on delivery. Results showed that of all formulations and cells tested, dual-ligand liposomes with equal ligand valencies achieved enhanced binding and selectivity for cancer cells expressing equal and high levels of receptor expression. These trends were consistent between theoretical and experimental results. The optimized liposomes were further used to achieve efficient and selective transfection in dual-receptor expressing cancer cells. With a quantitative understanding of dual-ligand liposome binding, the insights gained from this study can inform rational design of modular heteromultivalent nanoparticles for enhanced specificity to target tissue for the creation of more effective cancer treatments.
Subject(s)
Integrin alpha5beta1/metabolism , Integrin alpha6beta4/metabolism , Peptides/chemistry , Animals , Cell Line, Tumor , Cell Survival , Gene Transfer Techniques , Humans , Ligands , Liposomes , Mice , Models, Biological , Nanoparticles , Particle Size , Peptides/metabolism , Plasmids , Polyethylene Glycols/chemistry , Rats , Surface PropertiesABSTRACT
Liposomal nanomedicine has led to clinically useful cancer therapeutics like Doxil and DaunoXome. In addition, peptide-functionalized liposomes represent an effective drug and gene delivery vehicle with increased cancer cell specificity, enhanced tumor-penetrating ability and high tumor growth inhibition. The goal of this article is to review the recently published literature of the peptide-amphiphiles that were used to functionalize liposomes, to highlight successful designs that improved drug and gene delivery to cancer cells in vitro, and cancer tumors in vivo, and to discuss the current challenges of designing these peptide-decorated liposomes for effective cancer treatment.
Subject(s)
Antineoplastic Agents/administration & dosage , Drug Delivery Systems/methods , Drug Design , Gene Transfer Techniques , Neoplasms/drug therapy , Neoplasms/genetics , Peptides/chemistry , Animals , Humans , Ligands , Liposomes , Neoplasms/metabolism , Peptides/chemical synthesisABSTRACT
A set of poly(δ-valerolactone-co-d,l-lactide)-b-poly(ethylene glycol)-b-poly(δ-valerolactone-co-d,l-lactide) (PVLA-PEG-PVLA) triblock copolymers was synthesized and the solution properties were characterized using rheology, cryo-TEM, cryo-SEM, SANS, and degradation studies. This polymer self-assembles into a low viscosity fluid with flowerlike spherical micelles in water at room temperature and transforms into a wormlike morphology upon heating, accompanied by gelation. At even higher temperatures syneresis is observed. At physiological temperature (37 °C) the hydrogel's average pore size is around 600 nm. The PVLA-PEG-PVLA gel degrades in about 45 days in cell media, making this unique hydrogel a promising candidate for biomedical applications.
ABSTRACT
UNLABELLED: Tissue organoids are a promising technology that may accelerate development of the societal and NIH mandate for precision medicine. Here we describe a robust and simple method for generating cerebral organoids (cOrgs) from human pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. By using no additional neural induction components, cOrgs appeared on the hydrogel surface within 10-14 days, and under static culture conditions, they attained sizes up to 3 mm in greatest dimension by day 28. Histologically, the organoids showed neural rosette and neural tube-like structures and evidence of early corticogenesis. Immunostaining and quantitative reverse-transcription polymerase chain reaction demonstrated protein and gene expression representative of forebrain, midbrain, and hindbrain development. Physiologic studies showed responses to glutamate and depolarization in many cells, consistent with neural behavior. The method of cerebral organoid generation described here facilitates access to this technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable. SIGNIFICANCE: Tissue organoids are a promising technology with many potential applications, such as pharmaceutical screens and development of in vitro disease models, particularly for human polygenic conditions where animal models are insufficient. This work describes a robust and simple method for generating cerebral organoids from human induced pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. This method, by virtue of its simplicity and use of defined materials, greatly facilitates access to cerebral organoid technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.