Cationic Nanohydrogel Particles as Potential siRNA Carriers for Cellular Delivery

Oligonucleotides such as short, double-stranded RNA (siRNA) or plasmid DNA (pDNA) promise high potential in gene therapy. For pharmaceutical application, however, adequate drug carriers are required. Among various concepts progressing in the market or final development, nanosized hydrogel particles may serve as novel transport media especially for siRNA. In this work, a new concept of synthesizing polymeric cationic nanohydrogels was developed, which offers a promising strategy to complex and transport siRNA into cells. For this purpose, amphiphilic reactive ester block copolymers were synthesized by RAFT polymerization of pentafluorophenyl methacrylate as reactive ester monomer together wi…

A fully synthetic glycopeptide antitumor vaccine based on multiple antigen presentation on a hyperbranched polymer.

For antitumor vaccines both the selected tumor-associated antigen, as well as the mode of its presentation, affect the immune response. According to the principle of multiple antigen presentation, a tumor-associated MUC1 glycopeptide combined with the immunostimulating T-cell epitope P2 from tetanus toxoid was coupled to a multi-functionalized hyperbranched polyglycerol by "click chemistry". This globular polymeric carrier has a flexible dendrimer-like structure, which allows optimal antigen presentation to the immune system. The resulting fully synthetic vaccine induced strong immune responses in mice and IgG antibodies recognizing human breast-cancer cells.

Not just for tumor targeting: unmet medical needs and opportunities for nanomedicine.

During the last 3 decades, nanomedicines have provided novel opportunities to improve the delivery of chemotherapeutics in cancer therapy effectively. However, many principles learnt from there have the potential to be transferred to other diseases. This perspective article, on the one hand, critically reflects the limitations of nanomedicines in tumor therapy and, on the other hand, provides alternative examples of nanomedicinal applications in immunotherapy, noninvasive drug deliveries across epithelial barriers and strategies to combat intra- and extra-cellular bacterial infections. Looking ahead, access to highly complex nanoparticular delivery vehicles given nowadays may allow further…

Squaric Ester-Based, pH-Degradable Nanogels: Modular Nanocarriers for Safe, Systemic Administration of Toll-like Receptor 7/8 Agonistic Immune Modulators

Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which i…

New Perspectives of HPMA-based Copolymers Derived by Post-Polymerization Modification

Poly[N-(2-hydroxypropyl) methacrylamide] (HPMA) was one of the first polymers applied as polymer drug conjugate in the clinics. Since then many attempts have been made to expand the functionality of HPMA-based copolymers from advanced synthetic pathways to multiple biomedical applications. This Feature Article highlights multifunctional HPMA based copolymers prepared by controlled radical polymerization and subsequent post-polymerization modification of activated ester precursor polymers via aminolysis. This approach combines precise control of the polymer's microstructure (molecular weight, dispersity, block copolymer formation, end group functionalization) with an easy introduction of var…

Cationic Nanohydrogel Particles for Therapeutic Oligonucleotide Delivery.

Short pharmaceutical active oligonucleotides such as small interfering RNA (siRNA) or cytidine-phosphate-guanosine (CpG) are considered as powerful therapeutic alternatives, especially to medicate hard-to-treat diseases (e.g., liver fibrosis or cancer). Unfortunately, these molecules are equipped with poor pharmacokinetic properties that prevent them from translation. Well-defined nanosized carriers can provide opportunities to optimize their delivery and guide them to their site of action. Among several concepts, this Feature Article focuses on cationic nanohydrogel particles as a universal delivery system for small anionic molecules including siRNA and CpG. Cationic nanohydrogels are deri…

SiRNA-mediated in vivo gene knockdown by acid-degradable cationic nanohydrogel particles

Cationic nanohydrogel particles have become an attractive tool for systemic siRNA delivery, but improvement of their in vivo tolerance is desirable, especially to prevent potential long term side effects by tissue and cellular accumulation. Here, we designed novel ketal cross-linked cationic nanohydrogel particles that were assessed for reduced tissue accumulation and robust siRNA delivery in vitro and in vivo. An oligo-amine cross-linker equipped with a ketal moiety in its core was synthesized and applied to nanohydrogel cross-linking of self-assembled reactive ester block copolymers in DMSO. The resulting acid-sensitive cationic nanoparticles spontaneously disassembled over time in acidic…

Novel Opportunities for Cathepsin S Inhibitors in Cancer Immunotherapy by Nanocarrier-Mediated Delivery

Cathepsin S (CatS) is a secreted cysteine protease that cleaves certain extracellular matrix proteins, regulates antigen presentation in antigen-presenting cells (APC), and promotes M2-type macrophage and dendritic cell polarization. CatS is overexpressed in many solid cancers, and overall, it appears to promote an immune-suppressive and tumor-promoting microenvironment. While most data suggest that CatS inhibition or knockdown promotes anti-cancer immunity, cell-specific inhibition, especially in myeloid cells, appears to be important for therapeutic efficacy. This makes the design of CatS selective inhibitors and their targeting to tumor-associated M2-type macrophages (TAM) and DC an attr…

Addressing Dendritic Cells for Anticancer Immunity

RAFT-polymerized poly(hexafluoroisopropyl methacrylate)s as precursors for functional water-soluble polymers

Post-polymerization modification of well-defined precursor polymers is a versatile tool to obtain multifunctional water-soluble polymers that cannot be synthesized by common polymerization techniques. For the first time, 1,1,1,3,3,3-hexafluoroisopropyl methacrylate (HFIPMA) based homo and block copolymers were synthesized via RAFT polymerization to provide precise precursors for the post-polymerization modification of the 1,1,1,3,3,3-hexafluoroisopropyl ester side chains with water-soluble amines (methoxy tri(ethylene glycol) amine, 2-hydroxypropyl amine and 3-(dimethylamino)-1-propylamine). Sequential aminolysis using Oregon Green cadaverine first followed by 2-hydroxypropyl amine enables …

Degradable cationic nanohydrogel particles for stimuli-responsive release of siRNA.

Well-defined nanogels have become quite attractive as safe and stable carriers for siRNA delivery. However, to avoid nanoparticle accumulation, they need to provide a stimuli-responsive degradation mechanism that can be activated at the payload's site of action. In this work, the synthetic concept for generating well-defined nanohydrogel particles is extended to incorporate disulfide cross-linkers into a cationic nanonetwork for redox-triggered release of oligonucleotide payload as well as nanoparticle degradation under reductive conditions of the cytoplasm. Therefore, a novel disulfide-modified spermine cross-linker is designed that both allows disassembly of the nanogel as well as removal…

Linear-Hyperbranched Graft-Copolymers via Grafting-to Strategy Based on Hyperbranched Dendron Analogues and Reactive Ester Polymers

The synthesis of hyperbranched polyglycerol dendron analogues with precisely one focal amino functionality (H2N-hbPG) and their use for the synthesis of linear-hyperbranched graft-copolymers in a grafting-to approach is reported. By use of N,N-dibenzyl tris(hydroxylmethyl) aminomethane as a novel initiator for the ring-opening multibranching polymerization of glycidol, dendron analogues with one focal amino functionality of molecular weights ranging from 500 to 15000 g mol–1 and narrow to moderate polydispersities (Mw/Mn = 1.2–1.9) were synthesized, as confirmed by NMR and SEC. After removal of the benzyl protective groups, the accessibility and selective transformation of the focal amino g…

Aggregation behavior of cationic nanohydrogel particles in human blood serum.

For systemic siRNA delivery applications, well-defined drug carriers are required that guarantee stability for both carrier and cargo. Among various concepts progressing in market or final development, cationic nanohydrogel particles may serve as novel transport media especially designed for siRNA-in vivo experiments. In this work, the interaction of nanohydrogel particles with proteins and serum components was studied via dynamic light scattering in human blood serum as novel screening method prior to applications in vivo. The formation of larger aggregates mostly caused by charge interaction with albumin could be suppressed by nanogel loading with siRNA affording a neutral zeta potential …

Size-dependent knockdown potential of siRNA-loaded cationic nanohydrogel particles.

To overcome the poor pharmacokinetic conditions of short double-stranded RNA molecules in RNA interference therapies, cationic nanohydrogel particles can be considered as alternative safe and stable carriers for oligonucleotide delivery. For understanding key parameters during this process, two different types of well-defined cationic nanohydrogel particles were synthesized, which provided nearly identical physicochemical properties with regards to their material composition and resulting siRNA loading characteristics. Yet, according to the manufacturing process using amphiphilic reactive ester block copolymers of pentafluorophenyl methacrylate (PFPMA) and tri(ethylene glycol)methyl ether m…

Reductive Decationizable Block Copolymers for Stimuli-Responsive mRNA Delivery

Messenger ribonucleic acids (mRNAs) are considered as promising alternatives for transient gene therapy, but to overcome their poor pharmacokinetic properties, smart carriers are required for cellular uptake and stimuli-responsive release. In this work, a synthetic concept toward reductive decationizable cationic block copolymers for mRNA complexation is introduced. By combination of RAFT block copolymerization with postpolymerization modification, cationic block copolymers are generated with disulfide-linked primary amines. They allow effective polyplex formation with negatively charged mRNA and subsequent release under reductive conditions of the cytoplasm. In first in vitro experiments w…

The Protein Corona as a Confounding Variable of Nanoparticle-Mediated Targeted Vaccine Delivery

Nanocarriers (NC) are very promising tools for cancer immunotherapy. Whereas conventional vaccines are based on the administration of an antigen and an adjuvant in an independent fashion, nanovaccines can facilitate cell-specific co-delivery of antigen and adjuvant. Furthermore, nanovaccines can be decorated on their surface with molecules that facilitate target-specific antigen delivery to certain antigen-presenting cell types or tumor cells. However, the target cell-specific uptake of nanovaccines is highly dependent on the modifications of the nanocarrier itself. One of these is the formation of a protein corona around NC after in vivo administration, which may potently affect cell-speci…

Macromol. Rapid Commun. 24/2014

Supramolecular Linear-g-Hyperbranched Graft Polymers: Topology and Binding Strength of Hyperbranched Side Chains

Complex, reversible hyperbranched graft polymer topologies have been obtained by spontaneous self-assembly. Well-defined adamantyl- and β-cyclodextrin-functionalized polymers were employed to generate linear-g-(linear–hyperbranched) supramolecular graft terpolymers. For this purpose the synthesis of monoadamantyl-functionalized linear polyglycerols (Ada-linPG) and hyperbranched polyglycerols (Ada-hbPG) as well as poly(ethylene glycol)-block-linear polyglycerol (Ada-PEG-b-linPG) and poly(ethylene glycol)-block-hyperbranched poly(glycerol) (Ada-PEG-b-hbPG) block copolymers was established. Isothermal titration calorimetry (ITC) with β-cyclodextrin revealed a shielding effect of hyperbranched …

Nanomedicine: In Vivo Gene-Silencing in Fibrotic Liver by siRNA-Loaded Cationic Nanohydrogel Particles (Adv. Healthcare Mater. 18/2015)

Density of conjugated antibody determines the extent of Fc receptor dependent capture of nanoparticles by liver sinusoidal endothelial cells

Despite considerable progress in the design of multifunctionalized nanoparticles (NPs) that selectively target specific cell types, their systemic application often results in unwanted liver accumulation. The exact mechanisms for this general observation are still unclear. Here we asked whether the number of cell-targeting antibodies per NP determines the extent of NP liver accumulation and also addressed the mechanisms by which antibody-coated NPs are retained in the liver. We used polysarcosine-based peptobrushes (PBs), which in an unmodified form remain in the circulation for >24 h due to the absence of a protein corona formation and low unspecific cell binding, and conjugated them with …

P0419 : In vivo cell specific gene silencing in the liver using novel siRNA-loaded nanohydrogel particles

Tunable dynamic hydrophobic attachment of guest molecules in amphiphilic core–shell polymers

In this study, synthesis and dynamic properties of amphiphilic core–shell polymers are reported as monitored through their interaction with small amphiphilic molecules. Brush-like structures are formed with a hydrophobic core surrounded by a hydrophilic shell utilizing controlled radical addition–fragmentation chain transfer (RAFT) polymerization of macromonomers consisting of linear polyglycerol chains attached to alkylene methacrylate. Continuous wave electron paramagnetic resonance (CW EPR) spectroscopy is employed to study how the amphiphilic, paramagnetic spin probe 16-DSA (16-doxyl stearic acid) interacts with polymers of different alkylene chain lengths in their hydrophobic cores and…

Nanoparticular bisphosphonate to selectively target and repolarize liver macrophages for efficient anti-tumour responses

Back Cover: Macromol. Biosci. 5/2014

Combining Ring-Opening Multibranching and RAFT Polymerization: Multifunctional Linear–Hyperbranched Block Copolymers via Hyperbranched Macro-Chain-Transfer Agents

The synthesis of a hyperbranched macro-chain-transfer agent for RAFT polymerization of functional methacrylate or methacrylamide monomers was achieved by selectively attaching one single CTA onto hyperbranched polyglycerol dendron analogues. The combination of ring-opening multibranching polymerization of glycidol and subsequent RAFT polymerization of the hyperbranched macro-chain-transfer agents created a new route to a variety of multifunctional linear–hyperbranched block topologies. All linear–hyperbranched block copolymers could be synthesized with controlled molecular weight (Mn = 3.2–43.7 kg/mol) and low polydispersity (PDI = 1.15–1.34). As first examples for this universal approach, …

Water-Soluble Polymers Coupled with Glycopeptide Antigens and T-Cell Epitopes as Potential Antitumor Vaccines

Highly decorated: Tumor-associated MUC1 glycopeptide and tetanus toxoid T-cell epitope P2 can be attached to water-soluble poly(N-(2-hydroxypropyl)methacrylamide) carriers by orthogonal ligation techniques. Fully synthetic vaccine A with additional nanostructure-promoting domains induced antibodies that exhibit high affinity to tumor cells.

In Vivo siRNA Delivery to Immunosuppressive Liver Macrophages by alpha-Mannosyl-Functionalized Cationic Nanohydrogel Particles

Macrophages are the front soldiers of the innate immune system and are vital for immune defense, tumor surveillance, and tissue homeostasis. In chronic diseases, including cancer and liver fibrosis, macrophages can be forced into an immunosuppressive and profibrotic M2 phenotype. M2-type macrophages overexpress the mannose receptor CD206. Targeting these cells via CD206 and macrophage repolarization towards an immune stimulating and antifibrotic M1 phenotype through RNA interference represents an appealing therapeutic approach. We designed nanohydrogel particles equipped with mannose residues on the surface (ManNP) that delivered siRNA more efficiently to M2 polarized macrophages compared t…

In Vivo Gene-Silencing in Fibrotic Liver by siRNA-Loaded Cationic Nanohydrogel Particles

Cationic nanohydrogel particles loaded with anti-Col1α1 siRNA suppress collagen synthesis and deposition in fibrotic mice: Systemically administered 40 nm sized nanogel particles accumulate in collagen-expressing cells in the liver. Their siRNA payload induces a sequence specific in vivo gene knockdown affording an efficient antifibrotic effect in mice with liver fibrosis.

In vivo gene silencing in the liver: Comparison of siRNA-loaded non biodegradable vs. biodegradable nanohydrogel particles for antifibrotic therapy

Enhancing Gene Knockdown Efficiencies by Comparing siRNA-Loaded Cationic Nanogel Particles of Different Sizes

lt;pgt;In order to silence the expression levels of pathogenic genes, small interfering RNA (siRNA) requires a nano-sized carrier for its safe and stable delivery into cells. In this research highlight, we focus on well-defined cationic nanohydrogel particles developed in our group for such purposes. To investigate the nanogels’ mechanism for enhanced knockdown efficiencies, we recently synthesized two sets of particles with similar material composition and siRNA-loading characteristics, but – according to the manufacturing process – of different sizes. Within this study, 100-nm-sized nanogel particles loaded with siRNA accumulated inside the lysosomes already after 4 h and could not induce…

Targeted Repolarization of Tumor‐Associated Macrophages via Imidazoquinoline‐Linked Nanobodies

Abstract Tumor‐associated macrophages (TAMs) promote the immune suppressive microenvironment inside tumors and are, therefore, considered as a promising target for the next generation of cancer immunotherapies. To repolarize their phenotype into a tumoricidal state, the Toll‐like receptor 7/8 agonist imidazoquinoline IMDQ is site‐specifically and quantitatively coupled to single chain antibody fragments, so‐called nanobodies, targeting the macrophage mannose receptor (MMR) on TAMs. Intravenous injection of these conjugates result in a tumor‐ and cell‐specific delivery of IMDQ into MMRhigh TAMs, causing a significant decline in tumor growth. This is accompanied by a repolarization of TAMs to…

Transient Multivalent Nanobody Targeting to CD206-Expressing Cells via PH-Degradable Nanogels

To target nanomedicines to specific cells, especially of the immune system, nanobodies can be considered as an attractive tool, as they lack the Fc part as compared to traditional antibodies and, thus, prevent unfavorable Fc-receptor mediated mistargeting. For that purpose, we have site-specifically conjugated CD206/MMR-targeting nanobodies to three types of dye-labeled nanogel derivatives: non-degradable nanogels, acid-degradable nanogels (with ketal crosslinks), and single polymer chains (also obtained after nanogel degradation). All of them can be obtained from the same reactive ester precursor block copolymer. After incubation with na&iuml

CpG-Loaded Multifunctional Cationic Nanohydrogel Particles as Self-Adjuvanting Glycopeptide Antitumor Vaccines

Self-adjuvanting antitumor vaccines by multifunctional cationic nanohydrogels loaded with CpG. A conjugate consisting of tumor-associated MUC1-glycopeptide B-cell epitope and tetanus toxin T-cell epitope P2 is linked to cationic nanogels. Oligonucleotide CpG complexation enhances toll-like receptor (TLR) stimulated T-cell proliferation and rapid immune activation. This co-delivery promotes induction of specific MUC1-antibodies binding to human breast tumor cells without external adjuvant.

Mit Glycopeptid-Antigenen und T-Zell-Epitopen verknüpfte wasserlösliche Polymere als potenzielle Antitumor-Vakzine

In Vivo Myofibroblast Specific Gene Silencing in the Liver Using Novel Sirna-Loaded Biodegradable Nanohydrogel Particles

α-Mannosyl-Functionalized Cationic Nanohydrogel Particles for Targeted Gene Knockdown in Immunosuppressive Macrophages

Immunosuppressive M2 macrophages govern the immunophathogenic micromilieu in many severe diseases including cancer or fibrosis, thus, their re-polarization through RNA interference is a promising concept to support combinatorial therapies. For targeted siRNA delivery, however, safe and stable carriers are required that manage cell specific transport to M2 macrophages. Here, siRNA-loaded cationic nanogels are reported with α-mannosyl decorated surfaces that target and modify M2 macrophages selectively. Via amphiphilic precursor block copolymers bearing one single α-mannosyl moiety at their chain end mannosylated cationic nanohydrogel particles (ManNP) were obtained of 20 nm diameter determin…