Modulating efficacy and cytotoxicity of lipoamino fatty acid nucleic acid carriers using disulfide or hydrophobic spacers.

Double pH-responsive xenopeptides comprising polar ionizable succinoyl tetraethylene pentamine (Stp) motifs and lipophilic ionizable lipoamino fatty acids (LAFs) were recently found to efficiently transfect mRNA and pDNA at low doses. However, potency was often accompanied with cytotoxicity at higher doses. Insertion of bioreducible disulfide building blocks (ssbb) or non-reducible hydrophobic spacers between polar and apolar ionizable domains of LAF-Stp carriers should mitigate toxicity of xenopeptides. Carriers showed stable nucleic acid complexation and endosomal pH-dependent lytic activities, both of which were abolished after reductive cleavage of ssbb-containing carriers. For pDNA, U-shaped carriers with one Stp and two LAF units or bundle carriers with two Stps and four LAFs displayed highest potency. For mRNA, best transfection was achieved with bundle carriers with one Stp and four LAFs. Both the ssbb and hydrophobic spacer containing analogs displayed improved metabolic activity, reduced membrane damage, and improved cell growth. The ssbb carriers were most beneficial regarding living cell count and low apoptosis rates. Mechanistically, inserted spacers decelerated the transfection kinetics and altered the requirement of endosomal protonation. Overall, mRNA and pDNA carriers with improved biocompatibility have been designed, with their high potency illustrated in transfection of various cell lines including low passage number colon carcinoma cells.

In Vivo Endothelial Cell Gene Silencing by siRNA-LNPs Tuned with Lipoamino Bundle Chemical and Ligand Targeting.

Although small-interfering RNAs (siRNAs) are specific silencers for numerous disease-related genes, their clinical applications still require safe and effective means of delivery into target cells. Highly efficient lipid nanoparticles (LNPs) are developed for siRNA delivery, showcasing the advantages of novel pH-responsive lipoamino xenopeptide (XP) carriers. These sequence-defined XPs are assembled by branched lysine linkages between cationizable polar succinoyl tetraethylene pentamine (Stp) units and apolar lipoamino fatty acids (LAFs) at various ratios into bundle or U-shape topologies. Formulation of siRNA-LNPs using LAF4-Stp1 XPs as ionizable compounds led to robust cellular uptake, high endosomal escape, and successful in vitro gene silencing activity at an extremely low (150 picogram) siRNA dose. Of significance is the functional in vivo endothelium tropism of siRNA-LNPs with bundle LAF4-Stp1 XP after intravenous injection into mice, demonstrated by superior knockdown of liver sinusoidal endothelial cell (LSEC)-derived factor VIII (FVIII) and moderate silencing of hepatocyte-derived FVII compared to DLin-MC3-DMA-based LNPs. Optimizing lipid composition following click-modification of siRNA-LNPs with ligand c(RGDfK) efficiently silenced vascular endothelial growth factor receptor-2 (VEGFR-2) in tumor endothelial cells (TECs). The findings shed light on the role of ionizable XPs in the LNP in vivo cell-type functional targeting, laying the groundwork for future therapeutic applications.

Dual Effect by Chemical Electron Transfer Enhanced siRNA Lipid Nanoparticles: Reactive Oxygen Species-Triggered Tumor Cell Killing Aggravated by Nrf2 Gene Silencing.

Insufficient endosomal escape presents a major hurdle for successful nucleic acid therapy. Here, for the first time, a chemical electron transfer (CET) system was integrated into small interfering RNA (siRNA) lipid nanoparticles (LNPs). The CET acceptor can be chemically excited using the generated energy between the donor and hydrogen peroxide, which triggers the generation of reactive oxygen species (ROS), promoting endosomal lipid membrane destabilization. Tetra-oleoyl tri-lysino succinoyl tetraethylene pentamine was included as an ionizable lipopeptide with a U-shaped topology for effective siRNA encapsulation and pH-induced endosomal escape. LNPs loaded with siRNA and CET components demonstrated a more efficient endosomal escape, as evidenced by a galectin-8-mRuby reporter; ROS significantly augmented galectin-8 recruitment by at least threefold compared with the control groups, with a p value of 0.03. Moreover, CET-enhanced LNPs achieved a 24% improvement in apoptosis level by knocking down the tumor-protective gene nuclear factor erythroid 2-related factor 2, boosting the CET-mediated ROS cell killing.

Lipo-Xenopeptide Polyplexes for CRISPR/Cas9 based Gene editing at ultra-low dose.

Double pH-responsive xenopeptide carriers containing succinoyl tetraethylene pentamine (Stp) and lipo amino fatty acids (LAFs) were evaluated for CRISPR/Cas9 based genome editing. Different carrier topologies, variation of LAF/Stp ratios and LAF types as Cas9 mRNA/sgRNA polyplexes were screened in three different reporter cell lines using three different genomic targets (Pcsk9, eGFP, mdx exon 23). One U-shaped and three bundle (B2)-shaped lipo-xenopeptides exhibiting remarkable efficiencies were identified. Genome editing potency of top carriers were observed at sub-nanomolar EC50 concentrations of 0.4 nM sgRNA and 0.1 nM sgRNA for the top U-shape and top B2 carriers, respectively, even after incubation in full (≥ 90%) serum. Polyplexes co-delivering Cas9 mRNA/sgRNA with a single stranded DNA template for homology directed gene editing resulted in up to 38% conversion of eGFP to BFP in reporter cells. Top carriers were formulated as polyplexes or lipid nanoparticles (LNPs) for subsequent in vivo administration. Formulations displayed long-term physicochemical and functional stability upon storage at 4 °C. Importantly, intravenous administration of polyplexes or LNPs mediated in vivo editing of the dystrophin gene, triggering mRNA exon 23 splicing modulation in dystrophin-expressing cardiac muscle, skeletal muscle and brain tissue.

GalNAc- or Mannose-PEG-Functionalized Polyplexes Enable Effective Lectin-Mediated DNA Delivery.

A cationic, dendrimer-like oligo(aminoamide) carrier with four-arm topology based on succinoyl tetraethylene pentamine and histidines, cysteines, and N-terminal azido-lysines was screened for plasmid DNA delivery on various cell lines. The incorporated azides allow modification with various shielding agents of different polyethylene glycol (PEG) lengths and/or different ligands by copper-free click reaction, either before or after polyplex formation. Prefunctionalization was found to be advantageous over postfunctionalization in terms of nanoparticle formation, stability, and efficacy. A length of 24 ethylene oxide repetition units and prefunctionalization of ≥50% of azides per carrier promoted optimal polyplex shielding. PEG shielding resulted in drastically reduced DNA transfer, which could be successfully restored by active lectin targeting via novel GalNAc or mannose ligands, enabling enhanced receptor-mediated endocytosis of the carrier system. The involvement of the asialoglycoprotein receptor (ASGPR) in the uptake of GalNAc-functionalized polyplexes was confirmed in the ASGPR-positive hepatocarcinoma cell lines HepG2 and Huh7. Mannose-modified polyplexes showed superior cellular uptake and transfection efficacy compared to unmodified and shielded polyplexes in mannose-receptor-expressing dendritic cell-like DC2.4 cells.

Lipoamino bundle LNPs for efficient mRNA transfection of dendritic cells and macrophages show high spleen selectivity.

Messenger RNA (mRNA) is a powerful tool for nucleic acid-based therapies and vaccination, but efficient and specific delivery to target tissues remains a significant challenge. In this study, we demonstrate lipoamino xenopeptide carriers as components of highly efficient mRNA LNPs. These lipo-xenopeptides are defined as 2D sequences in different 3D topologies (bundles or different U-shapes). The polar artificial amino acid tetraethylene pentamino succinic acid (Stp) and various lipophilic tertiary lipoamino fatty acids (LAFs) act as ionizable amphiphilic units, connected in different ratios via bisamidated lysines as branching units. A series of more lipophilic LAF4-Stp1 carriers with bundle topology is especially well suited for efficient encapsulation of mRNA into LNPs, facilitated cellular uptake and strongly enhanced endosomal escape. These LNPs display improved, faster transfection kinetics compared to standard LNP formulations, with high potency in a variety of tumor cell lines (including N2a neuroblastoma, HepG2 and Huh7 hepatocellular, and HeLa cervical carcinoma cells), J774A.1 macrophages, and DC2.4 dendritic cells. High transfection levels were obtained even in the presence of serum at very low sub-microgram mRNA doses. Upon intravenous application of only 3 µg mRNA per mouse, in vivo mRNA expression is found with a high selectivity for dendritic cells and macrophages, resulting in a particularly high overall preferred expression in the spleen.

mCherry on Top: A Positive Read-Out Cellular Platform for Screening DMD Exon Skipping Xenopeptide-PMO Conjugates.

Phosphorodiamidate morpholino oligomers (PMOs) are a special type of antisense oligonucleotides (ASOs) that can be used as therapeutic modulators of pre-mRNA splicing. Application of nucleic-acid-based therapeutics generally requires suitable delivery systems to enable efficient transport to intended tissues and intracellular targets. To identify potent formulations of PMOs, we established a new in vitro-in vivo screening platform based on mdx exon 23 skipping. Here, a new in vitro positive read-out system (mCherry-DMDEx23) is presented that is sensitive toward the PMO(Ex23) sequence mediating DMD exon 23 skipping and, in this model, functional mCherry expression. After establishment of the reporter system in HeLa cells, a set of amphiphilic, ionizable xenopeptides (XPs) was screened in order to identify potent carriers for PMO delivery. The identified best-performing PMO formulation with high splice-switching activity at nanomolar concentrations in vitro was then translated to in vivo trials, where exon 23 skipping in different organs of healthy BALB/c mice was confirmed. The predesigned in vitro-in vivo workflow enables evaluation of PMO(Ex23) carriers without change of the PMO sequence and formulation composition. Furthermore, the identified PMO-XP conjugate formulation was found to induce highly potent exon skipping in vitro and redistributed PMO activity in different organs in vivo.

Peptide nucleic acid-zirconium coordination nanoparticles.

Ideal drug carriers feature a high loading capacity to minimize the exposure of patients with excessive, inactive carrier materials. The highest imaginable loading capacity could be achieved by nanocarriers, which are assembled from the therapeutic cargo molecules themselves. Here, we describe peptide nucleic acid (PNA)-based zirconium (Zr) coordination nanoparticles which exhibit very high PNA loading of [Formula: see text] w/w. This metal-organic hybrid nanomaterial class extends the enormous compound space of coordination polymers towards bioactive oligonucleotide linkers. The architecture of single- or double-stranded PNAs was systematically varied to identify design criteria for the coordination driven self-assembly with Zr(IV) nodes at room temperature. Aromatic carboxylic acid functions, serving as Lewis bases, and a two-step synthesis process with preformation of [Formula: see text] turned out to be decisive for successful nanoparticle assembly. Confocal laser scanning microscopy confirmed that the PNA-Zr nanoparticles are readily internalized by cells. PNA-Zr nanoparticles, coated with a cationic lipopeptide, successfully delivered an antisense PNA sequence for splicing correction of the [Formula: see text]-globin intron mutation IVS2-705 into a functional reporter cell line and mediated splice-switching via interaction with the endogenous mRNA splicing machinery. The presented PNA-Zr nanoparticles represent a bioactive platform with high design flexibility and extraordinary PNA loading capacity, where the nucleic acid constitutes an integral part of the material, instead of being loaded into passive delivery systems.

Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery.

The introduction of the CRISPR/Cas9 system in the form of Cas9/sgRNA ribonucleoproteins (RNP) is an efficient, straightforward strategy for genome editing, and potent RNP carriers are in high demand. Here, we report a series of artificial peptides based on novel ionizable amino acids that are able to deliver Cas9 RNP into cells very efficiently. Systematic variation of hydrophobic properties revealed a relationship between the xenopeptide logD7.4 and genome editing potency. By correlating the physicochemical properties with biological activity, individual optima were found for different xenopeptide sequence architectures. The optimized amphiphilic carriers enable ∼88% eGFP knockout at an RNP dose of only 1 nM and up to 40% homology-directed repair (HDR) in eGFP/BFP switchable reporter cells by co-delivery with an ssDNA template. Mechanistic studies demonstrated that hydrophobically balanced xenopeptides are more resistant to ionic stress as well as concentration-dependent dissociation and promote endocytosis by both clathrin- and macropinocytosis-mediated pathways. The systematic study develops a versatile and adjustable carrier platform and highlights impactful structure-activity relationships, providing a new chemical guide for the design and optimization of nonviral Cas9 RNP nanocarriers.

Iron-Gallic Acid Peptide Nanoparticles as a Versatile Platform for Cellular Delivery with Synergistic ROS Enhancement Effect.

Cytosolic delivery of peptides is of great interest owing to their biological functions, which could be utilized for therapeutic applications. However, their susceptibility to enzymatic degradation and multiple cellular barriers generally hinders their clinical application. Integration into nanoparticles, which can enhance the stability and membrane permeability of bioactive peptides, is a promising strategy to overcome extracellular and intracellular obstacles. Herein, we present a versatile platform for the cellular delivery of various cargo peptides by integration into metallo-peptidic coordination nanoparticles. Both termini of cargo peptides were conjugated with gallic acid (GA) to assemble GA-modified peptides into nanostructures upon coordination of Fe(III). Initial pre-complexation of Fe(III) by poly-(vinylpolypyrrolidon) (PVP) as a template favored the formation of nanoparticles, which are able to deliver the peptides into cells efficiently. Iron-gallic acid peptide nanoparticles (IGPNs) are stable in water and are supposed to generate reactive oxygen species (ROS) from endogenous H2O2 in cells via the Fenton reaction. The strategy was successfully applied to an exemplary set of peptide sequences varying in length (1-7 amino acids) and charge (negative, neutral, positive). To confirm the capability of transporting bioactive cargos into cells, pro-apoptotic peptides were integrated into IGPNs, which demonstrated potent killing of human cervix carcinoma HeLa and murine neuroblastoma N2a cells at a 10 µM peptide concentration via the complementary mechanisms of peptide-triggered apoptosis and Fe(III)-mediated ROS generation. This study demonstrates the establishment of IGPNs as a novel and versatile platform for the assembly of peptides into nanoparticles, which can be used for cellular delivery of bioactive peptides combined with intrinsic ROS generation.

Molecular Chameleon Carriers for Nucleic Acid Delivery: The Sweet Spot between Lipoplexes and Polyplexes.

Taking advantage of effective intracellular delivery mechanisms of both cationizable lipids and polymers, highly potent double pH-responsive nucleic acid carriers are generated by combining at least two lipo amino fatty acids (LAFs) as hydrophobic cationizable motifs with hydrophilic cationizable aminoethylene units into novel sequence-defined molecules. The pH-dependent tunable polarity of the LAF is successfully implemented by inserting a central tertiary amine, which disrupts the hydrophobic character once protonated, resulting in pH-dependent structural and physical changes. This "molecular chameleon character" turns out to be advantageous for dynamic nucleic acid delivery via lipopolyplexes. By screening different topologies (blocks, bundles, T-shapes, U-shapes), LAF types, and LAF/aminoethylene ratios, highly potent pDNA, mRNA, and siRNA carriers are identified, which are up to several 100-fold more efficient than previous carrier generations and characterized by very fast transfection kinetics. mRNA lipopolyplexes maintain high transfection activity in cell culture even in the presence of ≥90% serum at an ultra-low mRNA dose of 3 picogram (≈2 nanoparticles/cell), and thus are comparable in potency to viral nanoparticles. Importantly, they show great in vivo performance with high expression levels especially in spleen, tumor, lungs, and liver upon intravenous administration of 1-3 µg luciferase-encoding mRNA in mice.

Folate Receptor-Mediated Delivery of Cas9 RNP for Enhanced Immune Checkpoint Disruption in Cancer Cells.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system offers great opportunities for the treatment of numerous diseases by precise modification of the genome. The functional unit of the system is represented by Cas9/sgRNA ribonucleoproteins (RNP), which mediate sequence-specific cleavage of DNA. For therapeutic applications, efficient and cell-specific transport into target cells is essential. Here, Cas9 RNP nanocarriers are described, which are based on lipid-modified oligoamino amides and folic acid (FolA)-PEG to realize receptor-mediated uptake and gene editing in cancer cells. In vitro studies confirm strongly enhanced potency of receptor-mediated delivery, and the nanocarriers enable efficient knockout of GFP and two immune checkpoint genes, PD-L1 and PVR, at low nanomolar concentrations. Compared with non-targeted nanoparticles, FolA-modified nanocarriers achieve substantially higher gene editing including dual PD-L1/PVR gene disruption after injection into CT26 tumors in vivo. In the syngeneic mouse model, dual disruption of PD-L1 and PVR leads to CD8+ T cell recruitment and distinct CT26 tumor growth inhibition, clearly superior to the individual knockouts alone. The reported Cas9 RNP nanocarriers represent a versatile platform for potent and receptor-specific gene editing. In addition, the study demonstrates a promising strategy for cancer immunotherapy by permanent and combined immune checkpoint disruption.

Receptor-Targeted Dual pH-Triggered Intracellular Protein Transfer.

Protein therapeutics are of widespread interest due to their successful performance in the current pharmaceutical and medical fields, even though their broad applications have been hindered by the lack of an efficient intracellular delivery approach. Herein, we fabricated an active-targeted dual pH-responsive delivery system with favorable tumor cell entry augmented by extracellular pH-triggered charge reversal and tumor receptor targeting and pH-controlled endosomal release in a traceless fashion. As a traceable model protein, the enhanced green fluorescent protein (eGFP) bearing a nuclear localization signal was covalently coupled with a pH-labile traceless azidomethyl-methylmaleic anhydride (AzMMMan) linker followed by functionalization with different molar equivalents of two dibenzocyclooctyne-octa-arginine-cysteine (DBCO-R8C)-modified moieties: polyethylene glycol (PEG)-GE11 peptide for epidermal growth factor receptor-mediated targeting and melittin for endosomal escape. The cationic melittin domain was masked with tetrahydrophthalic anhydride revertible at mild acidic pH 6.5. At the optimally balanced ratio of functional units, the on-demand charge conversion at tumoral extracellular pH 6.5 in combination with GE11-mediated targeting triggered enhanced electrostatic cellular attraction by the R8C cell-penetrating peptides and melittin, as demonstrated by strongly enhanced cellular uptake. Successful endosomal release followed by nuclear localization of the eGFP cargo was obtained by taking advantage of melittin-mediated endosomal escape and rapid traceless release from the AzMMMan linker. The effectiveness of this multifunctional bioresponsive system suggests a promising strategy for delivery of protein drugs toward intracellular targets. A possible therapeutic relevance was indicated by an example of cytosolic delivery of cytochrome c initiating the apoptosis pathway to kill cancer cells.

Hyaluronate siRNA nanoparticles with positive charge display rapid attachment to tumor endothelium and penetration into tumors.

A cationizable sequence-defined lipo-oligoaminoamide (lipo-OAA) conferring stable assembly of siRNA into ~200 nm sized complexes contains an N-terminal azidolysine for covalent coating of formed nanoparticles with dibenzocyclooctyne-amine (DBCO)-modified hyaluronic acid (HA). Depending on the applied equivalents of DBCO-HA, stable nanoparticles with either cationic or anionic surface charge can be formed. The unmodified and two types of covalent HA-modified siRNA nanoparticles differ in their biological characteristics. Both types of HA coated siRNA complexes show an enhanced cellular uptake over uncoated complexes and facilitate efficient gene silencing, but differ in intracellular uptake pathways and distribution. Upon intravenous administration in mice, beyond our expectation and in contrast to the in vitro findings, only the cationic HA nanoparticles but neither the non-coated cationic nor the anionic HA complexes were able to target subcutaneous Huh 7 tumors and exert potent (78%) gene silencing. The favorable and very fast accumulation of cationic HA nanoparticles was confirmed in another subcutaneous tumor model. As evidenced by 3D nanoparticle distribution within Huh 7 tumors evaluated at early time points of 5 min and 45 min, only the cationic HA-based nanoparticles rapidly attach to the tumor endothelium and subsequently penetrate into tumor, in contrast to the analogous anionic HA coated or the cationic non-coated formulation.

Delivery of Cas9/sgRNA Ribonucleoprotein Complexes via Hydroxystearyl Oligoamino Amides.

The programmable endonuclease activity and simple usage of CRISPR/Cas9 have revolutionized the field of genome editing. The binding of single guide RNA (sgRNA) by the Cas9 protein results in the formation of negatively charged ribonucleoprotein (RNP) complexes. The presence of this functional complex inside cells is imperative for the intended specific genome modifications. The direct intracellular delivery of Cas9/sgRNA RNP complexes is of great advantage. In this work, a compound library of sequence-defined oligo(ethylenamino) amides containing structural motifs for stable nanoparticle formation, cellular uptake, and endosomal release was used for the screening and development of suitable Cas9 RNP delivery vehicles. Lipid-containing oligoaminoamides (lipo-OAAs) were identified as the most efficient carriers for intracellular Cas9/sgRNA delivery and gene disruption. Fluorescence correlation spectroscopy measurements indicated that the lipo-OAAs only interact with sgRNA-loaded Cas9 protein, which suggests exclusive ionic interaction with the negatively charged RNPs. The type of contained fatty acid turned out to have a critical impact on the knock out efficiency: the presence of one hydroxy group in the fatty acid dramatically changes the properties and performance of the resulting Cas9/sgRNA lipo-OAA complexes. The lipo-OAA-containing hydroxy-stearic acid (OHSteA) was superior to the analogues with saturated or unsaturated fatty acids without hydroxylation; it formed smaller and more defined nanoparticles with Cas9/sgRNA and improved the cellular uptake and endosomal release, which altogether resulted in an increased nuclear association and the highest gene knock out levels. The efficient and adaptable delivery platform has high potential for the future development of therapeutics based on precise genome modifications.

Double Click-Functionalized siRNA Polyplexes for Gene Silencing in Epidermal Growth Factor Receptor-Positive Tumor Cells.

Sequence-defined lipo-oligomers generated via solid-phase assisted synthesis have been developed as siRNA delivery systems for RNA-interference (RNAi) based gene silencing. Here, novel siRNA lipo-polyplexes were established, which were postmodified with monovalent or bivalent DBCO-PEG24 agents terminated with peptide GE11 (YHWYGYTPQNVI) for epidermal growth factor receptor (EGFR)-targeted siRNA delivery into EGFR-positive tumor cells. Lipo-oligomers containing eight cationizable succinoyltetraethylene-pentamine (Stp) units mediated higher siRNA nanoparticle core stability than those containing four Stp units, and the incorporation of histidines for enhanced endosomal buffer capacity resulted in an improved gene silencing efficiency. Lipo-polyplexes modified with monovalent or bivalent PEG-GE11 via the copper-free click reaction possessed significantly enhanced cellular internalization and transfection efficiency in EGF receptor-positive human cervical KB and hepatoma Huh7 cells in comparison with the corresponding lipo-polyplexes shielded with PEG24 without targeting. Furthermore, modification with the bivalent DBCO-PEG24-GE11 ligand resulted in higher gene silencing efficiency than modification with the same equivalents of the monovalent DBCO-PEG24-GE11 ligand.

Co-delivery of pretubulysin and siEG5 to EGFR overexpressing carcinoma cells.

Small interfering RNA (siRNA) represents a new class of therapeutic agents. Its successful intracellular delivery is a major challenge. Lipo-oligomeric carriers can complex siRNA into lipopolyplexes and thus mediate its cellular uptake. In this study, siRNA against the kinesin related mRNA EG5 gene (siEG5) and the microtubule inhibitor pretubulysin (PT) were co-formulated into polyplexes using azide-containing lipo-oligomer 1198. Nanoparticles were further modified by click reaction using shielding agent DBCO-PEG or EGFR targeting peptide GE11 (DBCO-PEG-GE11). Polyplexes displayed efficient payload incorporation and homogenous particle sizes of 200 nm. The biological effects of the unmodified and surface-functionalized polyplexes were investigated. The successful GE11-mediated intracellular delivery of siRNA into the EGFR overexpressing KB and Huh7 cell lines facilitated potent silencing of an EGFP-luciferase reporter gene by GFP siRNA. Specific downregulation of EG5 mRNA by siEG5 resulted in the expected antitumoral activity. The combination formulation 1198 siEG5 + PT provided superior antitumoral activity over free PT and 1198 siEG5.

Combination Chemotherapy of L1210 Tumors in Mice with Pretubulysin and Methotrexate Lipo-Oligomer Nanoparticles.

In the current study, nanoparticles containing the antimetabolite drug methotrexate (MTX) and the novel tubulin-binding drug pretubulysin (PT) were developed for combination chemotherapy. Polyelectrolyte complexes were formed based on ∼20 nm cationic nanomicelles of lipo-oligomer 454 with the anionic MTX at the molar ratio of 3:1, resulting in spherical nanoparticles with sizes of 150 nm (454 MTX). Particle formation in the presence of PT, which also interacts with 454, resulted in coloaded micelle complexes (454 PT+MTX) of 170 nm as demonstrated by transmission electron microscopy and dynamic light scattering measurements. Both drugs were incorporated to a high extent (∼85% for MTX, ∼70% for PT). Nanoparticles were stable in up to 20% serum and physiological NaCl solution. Cellular internalization of 454 PT+MTX into L1210 leukemia and KB cervix carcinoma cells was determined by confocal light scattering microscopy. The antitumor activity of the drug combination PT+MTX in both cell lines was strongly increased by drug formulation with 454 with IC50 values of PT+MTX decreasing 11-fold from 0.22 nM to 19 pM on L1210 cells and 6-fold from 2.8 to 0.48 nM on KB cervix carcinoma cells. Systemic treatment of NMRI nu/nu mice bearing subcutaneous L1210 tumors with 454 PT+MTX nanoparticles resulted in a more effective delay of tumor growth in comparison to the free drug combination of PT+MTX without 454. Importantly, nanoparticle formulation of PT+MTX with 454 increased the survival of mice by more than 100% compared to that of the buffer treated group and more than 40% compared to that of the free drug group.

Coordinative Binding of Polymers to Metal-Organic Framework Nanoparticles for Control of Interactions at the Biointerface.

Metal-organic framework nanoparticles (MOF NPs) are of growing interest in diagnostic and therapeutic applications, and due to their hybrid nature, they display enhanced properties compared to more established nanomaterials. The effective application of MOF NPs, however, is often hampered by limited control of their surface chemistry and understanding of their interactions at the biointerface. Using a surface coating approach, we found that coordinative polymer binding to Zr- fum NPs is a convenient way for peripheral surface functionalization. Different polymers with biomedical relevance were assessed for the ability to bind to the MOF surface. Carboxylic acid and amine containing polymers turned out to be potent surface coatings and a modulator replacement reaction was identified as the underlying mechanism. The strong binding of polycarboxylates was then used to shield the MOF surface with a double amphiphilic polyglutamate-polysarcosine block copolymer, which resulted in an exceptional high colloidal stability of the nanoparticles. The effect of polymer coating on interactions at the biointerface was tested with regard to cellular association and protein binding, which has, to the best of our knowledge, never been discussed in literature for functionalized MOF NPs. We conclude that the applied approach enables a high degree of chemical surface confinement, which could be used as a universal strategy for MOF NP functionalization. In this way, the physicochemical properties of MOF NPs could be tuned, which allows for control over their behavior in biological systems.

Combined antitumoral effects of pretubulysin and methotrexate.

Pretubulysin (PT), a potent tubulin-binding antitumoral drug, and the well-established antimetabolite methotrexate (MTX) were tested separately or in combination (PT+MTX) for antitumoral activity in L1210 leukemia cells or KB cervix carcinoma cells in vitro and in vivo in NMRI-nu/nu tumor mouse models. In cultured L1210 cells, treatment with PT or MTX displays strong antitumoral effects in vitro, and the combination PT+MTX exceeds the effect of single drugs. PT also potently kills the MTX resistant KB cell line, without significant MTX combination effect. Cell cycle analysis reveals the expected arrest in G1/S by MTX and in G2/M by PT. In both cell lines, the PT+MTX combination induces a G2/M arrest which is stronger than the PT-triggered G2/M arrest. PT+MTX does not change rates of apoptotic L1210 or KB cells as compared to single drug applications. Confocal laser scanning microscopy images show the microtubule disruption and nuclear fragmentation induced by PT treatment of L1210 and KB cells. MTX changes the architecture of the F-actin skeleton. PT+MTX combines the toxic effects of both drugs. In the in vivo setting, the antitumoral activity of drugs differs from their in vitro cytotoxicity, but their combination effects are more pronounced. MTX on its own does not display significant antitumoral activity, whereas PT reduces tumor growth in both L1210 and KB in vivo models. Consistent with the cell cycle effects, MTX combined at moderate dose boosts the antitumoral effect of PT in both in vivo tumor models. Therefore, the PT+MTX combination may present a promising therapeutic approach for different types of cancer.