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Translating innovative nanomaterials to medical products requires efficient manufacturing techniques that enable large-scale high-throughput synthesis and high reproducibility. Drug carriers in medicine embrace a complex subset of tasks calling for multi-functionality. Here, we report the synthesis of pro-drug-loaded core cross-linked polymeric micelles (CCPMs) in a continuous flow process, which combines the commonly separated steps of micelle formation, core cross-linking, functionalization, and purification into a single process. Redox-responsive CCPMs are formed from thiol-reactive polypept(o)ides of polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) and functional cross-linkers based on dihydrolipoic acid hydrazide for pH-dependent release of paclitaxel. The precisely controlled microfluidic self-assembly and purification process allows the production of spherical micelles (Dh = 35 nm) with low polydispersity values (PDI

Tuberculosis is the deadliest bacterial disease globally, threatening the lives of millions every year. New antibiotic therapies that can shorten the duration of treatment, improve cure rates, and impede the development of drug resistance are desperately needed. Here, we used polymeric micelles to encapsulate four second-generation derivatives of the antitubercular drug pretomanid that had previously displayed much better in vivo activity against Mycobacterium tuberculosis than pretomanid itself. Because these compounds were relatively hydrophobic, we expected that such micellar formulations would increase drug bioavailability, reduce toxicities, and improve therapeutic outcomes. The polymeric micelles were based on polypept(o)ides (PeptoMicelles) and were stabilized in their hydrophobic core by π-π interactions, allowing the efficient encapsulation of aromatic pretomanid derivatives. The stability of these π-π-stabilized PeptoMicelles was demonstrated in water, blood plasma, and lung surfactant by fluorescence cross-correlation spectroscopy and was further supported by prolonged circulation times of several days in the vasculature of zebrafish larvae. The pretomanid derivative with the best in vitro potency against Mycobacterium marinum (“drug D”) was also the most efficacious PeptoMicelle formulation tested in the zebrafish larvae infection model, almost completely eradicating the bacteria at non-toxic doses. This lead formulation was further assessed against Mycobacterium tuberculosis in the susceptible C3HeB/FeJ mouse model, which develops human-like necrotic granulomas. Following intravenous administration, the drug D micellar formulation significantly reduced bacterial burden and inflammatory responses in the lungs and spleens of infected mice.

We report on the synthesis of polysarcosine-block-poly(S-alkylsulfonyl)-l-cysteine block copolymers, which combine three orthogonal addressable groups enabling site-specific conversion of all reactive entities in a single step. The polymers are readily obtained by ring-opening polymerization (ROP) of corresponding α-amino acid N-carboxyanhydrides (NCAs) combining azide and amine chain ends, with a thiol-reactive S-alkylsulfonyl cysteine. Functional group interconversion of chain ends using strain-promoted azide–alkyne cycloaddition (SPAAC) and activated ester chemistry with NHS- and DBCO-containing fluorescent dyes could be readily performed without affecting the cross-linking reaction between thiols and S-alkylsulfonyl protective groups. Eventually, all three functionalities can be combined in the formation of multifunctional disulfide core cross-linked nanoparticles bearing spatially separated functionalities. The simultaneous attachment of dyes in core and corona during the formation of core-cross-link...

Secondary structure formation differentiates polypeptides from most of the other synthetic polymers, and the transitions from random coils to rod-like α-helices or β-sheets represent an additional parameter to direct self-assembly and the morphology of nanostructures. We investigated the influence of distinct secondary structures on the self-assembly of reactive amphiphilic polypept(o)ides. The individual morphologies can be preserved by core cross-linking via chemoselective disulfide bond formation. A series of thiol-responsive copolymers of racemic polysarcosine-block-poly(S-ethylsulfonyl-dl-cysteine) (pSar-b-p(dl)Cys), enantiopure polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) (pSar-b-p(l)Cys), and polysarcosine-block-poly(S-ethylsulfonyl-l-homocysteine) (pSar-b-p(l)Hcy) was prepared by N-carboxyanhydride polymerization. The secondary structure of the peptide segment varies from α-helices (pSar-b-p(l)Hcy) to antiparallel β-sheets (pSar-b-p(l)Cys) and disrupted β-sheets (pSar-b-p(dl)Cys). When subjected to nanoprecipitation, copolymers with antiparallel β-sheets display the strongest tendency to self-assemble, whereas disrupted β-sheets hardly induce aggregation. This translates to worm-like micelles, solely spherical micelles, or ellipsoidal structures, as analyzed by atomic force microscopy and cryogenic transmission electron microscopy, which underlines the potential of secondary structure-driven self-assembly of synthetic polypeptides.

Pretargeting is a powerful nuclear imaging strategy to achieve enhanced imaging contrast for nanomedicines. It reduces the radiation burden to healthy tissue. Pretargeting is based on bioorthogonal chemistry. The most attractive reaction for this purpose is currently the tetrazine ligation, which occurs between trans-cyclooctene (TCO) tags and tetrazines (Tzs). Pretargeted imaging beyond the blood-brain-barrier (BBB) has not been reported thus far. In this study, we developed Tz imaging agents that are capable to ligate in vivo to targets beyond the BBB. We chose to develop 18F-labeled Tzs as they can be applied to positron emission tomography (PET) - the most powerful molecular imaging technology. Fluorine-18 is an ideal radionuclide for PET due to its almost ideal decay properties. Fluorine-18 also allows - as a non-metal radionuclide - to develop Tzs with physicochemical properties enabling passive brain diffusion. In order to develop these imaging agents, we applied a rational drug design approach. This approach was based on estimated and experimental determined parameters such as the BBB score, pretargeted autoradiography contrast, in vivo input and washout curves as well as on metabolism studies. From initially 18 developed structures, five Tzs were selected to be tested on their in vivo click performance. Whereas all selected structures clicked in vivo into the brain, [18F]18 displayed the most favorable characteristics with respect to brain pretargeting. [18F]18 is our lead compound for future pretargeted imaging studies based on BBB-penetrant monoclonal antibodies. Pretargeting beyond the BBB will allow us to image targets beyond the BBB that are currently not imageable. For example, soluble protein isoforms could be imaged. These proteins are valuable drug targets for several neurodegenerative diseases and can currently not be imaged. Imaging would allow for diagnosis of these diseases, identifying responders from non-responders or to monitor treatment. Consequently, imaging will provide valuable information to accelerate drug development and greatly benefit patient care.

The use of nanoparticles as carriers is an extremely promising way for administration of therapeutic agents, such as drug molecules, proteins, and nucleic acids. Such nanocarriers (NCs) can increase the solubility of hydrophobic compounds, protect their cargo from the environment, and if properly functionalized, deliver it to specific target cells and tissues. Polymer-based NCs are especially promising, because they offer high degree of versatility and tunability. However, in order to get a full advantage of this therapeutic approach and develop efficient delivery systems, a careful characterization of the NCs is needed. This review highlights the fluorescence correlation spectroscopy (FCS) technique as a powerful and versatile tool for NCs characterization at all stages of the drug delivery process. In particular, FCS can monitor and quantify the size of the NCs and the drug loading efficiency after preparation, the NCs stability and possible interactions with, e.g., plasma proteins in the blood stream and the kinetic of drug release in the cytoplasm of the target cells.

The ring-opening polymerization (ROP) of N-carboxy anhydrides (NCAs) is mostly divided into two classes: NCAs of α-substituted amino acids and N-methylated NCAs of α-unsubstituted glycine derivatives (NNCAs). The use of both monomer types offers different mechanistic features and results in a multitude of functional materials. To combine these properties, the synthesis and ROP of α-substituted and N-methylated NCAs (αNNCAs) of several amino acids were investigated. The current study provides insight into the influence of polymerization conditions and the limitations caused by the enhanced steric demand of the amino acid NCA monomers and their N-methylated derivatives. Namely, the effects of solvent polarity (DMF and DCM) and steric demand of the initiator by using neopentyl amine (NPA) and n-butyl amine (nBu) were studied. Analysis by HFIP-GPC and MALDI-ToF MS reveals that the polymerization and the resulting polymers are tremendously affected by the steric demand of both the initiators and the monomers, while electronic effects seem to have only minor influences. The experimental results are further compared with computational studies, based on coupled cluster (CC) calculations, which underline that electronic effects are of lower importance than steric constraints for the ROP of αNNCAs. Moreover, poly(N-methyl-L-methionine) forms helical secondary structures in solution. Therefore, this work combines mechanistic studies of the ROP of αNNCAs with initial studies on the solution properties of these polypeptides.

A novel platform rationally integrating indocyanine green analogues and an arginine-rich dendritic peptide with both nitroreductase (NTR) and glutathione (GSH) reduction responsive linkers was developed. This multifunctional platform can enable selective and efficient gene delivery and specific turn-on fluorescence imaging in tumors.

ABC-type triblock copolymers are a rising platform especially for oligonucleotide delivery as they offer an additional functionality besides the anyhow needed functions of shielding and complexation. The authors present a polypept(o)ide-based triblock copolymer synthesized by amine-initiated ring-opening polymerization (ROP) of N-carboxyanhydrides (NCAs), comprising a shielding block A of polysarcosine (pSar), a poly(S-ethylsulfonyl-l-cystein) (pCys(SO2 Et)) block B for bioreversible and chemo-selective cross-linking and a poly(l-lysine) (pLys) block C for complexation to construct polyion complex (PIC) micelles as vehicle for small interfering RNA (siRNA) delivery. The self-assembly behavior of ABC-type triblocks is investigated to derive correlations between block lengths of the polymer and PIC micelle structure, showing an enormous effect of the β-sheet forming pCys(SO2 Et) block. Moreover, the block enables the introduction of disulfide cross-links by reaction with multifunctional thiols to increase stability against dilution. The right content of the additional block leads to well-defined cross-linked 50-60 nm PIC micelles purified from production impurities and determinable siRNA loading. These PIC micelles can deliver functional siRNA into Neuro2A and KB cells evaluated by cellular uptake and specific gene knockdown assays.

With the increasing emergence of drug-resistant Mycobacterium tuberculosis strains, new and effective antibiotics against tuberculosis (TB) are urgently needed. However, the high frequency of poorly water-soluble compounds among hits in high-throughput drug screening campaigns is a major obstacle in drug discovery. Moreover, in vivo testing using conventional animal TB models, such as mice, is time consuming and costly, and represents a major bottleneck in lead compound discovery and development. Here, we report the use of the zebrafish embryo TB model for evaluating the in vivo toxicity and efficacy of five poorly water-soluble nitronaphthofuran derivatives, which were recently identified as possessing anti-TB activity in vitro. To aid solubilization, compounds were formulated in biocompatible polymeric micelles (PMs). Three of the five PM-formulated nitronaphthofuran derivatives showed low toxicity in vivo, significantly reduced bacterial burden and improved survival in infected zebrafish embryos. We propose the zebrafish embryo TB-model as a quick and sensitive tool for evaluating the in vivo toxicity and efficacy of new anti-TB compounds during early stages of drug development. Thus, this model is well suited for pinpointing promising compounds for further development., Summary: The zebrafish embryo is a quick, sensitive and powerful tool for evaluating the in vivo toxicity and efficacy of newly identified lipophilic anti-tuberculosis compounds formulated in nanoparticles.

Introduction: Radiotherapy of cancer requires both alpha- and beta-particle emitting radionuclides, as these radionuclide types are efficient at destroying different types of tumors. Both classes of radionuclides require a vehicle, such as an antibody or a polymer, to be delivered and retained within the tumor. Polyglutamic acid (pGlu) is a polymer that has proven itself effective as a basis of drug-polymer conjugates in the clinic, while its derivatives have been used for pretargeted tumor imaging in a research setup. trans-Cyclooctene (TCO) modified pGlu is suitable for pretargeted imaging or therapy, as well as for intratumoral radionuclide therapy. In all cases, it becomes indirectly radiolabeled via the bioorthogonal click reaction with the tetrazine (Tz) molecule carrying the radionuclide. In this study, we report the radiolabeling of TCO-modified pGlu with either lutetium-177 (177Lu), a beta-particle emitter, or actinium-225 (225Ac), an alpha-particle emitter, using the click reaction between TCO and Tz. Methods: A panel of Tz derivatives containing a metal ion binding chelator (DOTA or macropa) connected to the Tz moiety directly or through a polyethylene glycol (PEG) linker was synthesized and tested for their ability to chelate 177Lu and 225Ac, and click to pGlu-TCO. Radiolabeled 177Lu-pGlu and 225Ac-pGlu were isolated by size exclusion chromatography. The retention of 177Lu or 225Ac by the obtained conjugates was investigated in vitro in human serum. Results: All DOTA-modified Tzs efficiently chelated 177Lu resulting in average radiochemical conversions (RCC) of >75%. Isolated radiochemical yields (RCY) for 177Lu-pGlu prepared from 177Lu-Tzs ranged from 31% to 55%. TLC analyses detected 177Lu for all 177Lu-pGlu preparations over six days in human serum. For 225Ac chelation, optimized RCCs ranged from 61 ± 34% to quantitative for DOTA-Tzs and were quantitative for the macropa-modified Tz (>98%). Isolated radiochemical yields (RCY) for 225Ac-pGlu prepared from 225Ac-Tzs ranged from 28% to 51%. For 3 out of 5 225Ac-pGlu conjugates prepared from DOTA-Tzs, the amount of unchelated 225Ac stayed below 10% over six days in human serum, while 225Ac-pGlu prepared from macropa-Tz showed a steady release of up to 37% 225Ac. Conclusion: We labeled TCO-modified pGlu polymers with alpha- and beta-emitting radionuclides in acceptable RCYs. All 177Lu-pGlu preparations and some 225Ac-pGlu preparations showed excellent stability in human plasma. Our work shows the potential of pGlu as a vehicle for alpha- and beta-radiotherapy of tumors and demonstrated the usefulness of Tz ligation for indirect radiolabeling.

Therapy resistance is the major cause of cancer death. As patients respond heterogeneously, precision/personalized medicine needs to be considered, including the application of nanoparticles (NPs). The success of therapeutic NPs requires to first identify clinically relevant resistance mechanisms and to define key players, followed by a rational design of biocompatible NPs capable to target resistance. Consequently, we employed a tiered experimental pipeline fromiin silico/ito analytical andiin vitro/ito overcome cisplatin resistance. First, we generated cisplatin-resistant cancer cells and used next-generation sequencing together with CRISPR/Cas9 knockout technology to identify the ion channel LRRC8A as a critical component for cisplatin resistance. LRRC8A's cisplatin-specificity was verified by testing free as well as nanoformulated paclitaxel or doxorubicin. The clinical relevance of LRRC8A was demonstrated by its differential expression in a cohort of 500 head and neck cancer patients, correlating with patient survival under cisplatin therapy. To overcome LRRC8A-mediated cisplatin resistance, we constructed cisplatin-loaded, polysarcosine-based core cross-linked polymeric NPs (NPsubCis/sub, Ø ∼ 28 nm) with good colloidal stability, biocompatibility (low immunogenicity, low toxicity, prolongediin vivo/icirculation, no complement activation, no plasma protein aggregation), and low corona formation properties. 2D/3D-spheroid cell models were employed to demonstrate that, in contrast to standard of care cisplatin, NPsubCis/subsignificantly (ip/ilt; 0.001) eradicated all cisplatin-resistant cells by circumventing the LRRC8A-transport pathwayivia/ithe endocytic delivery route. We here identified LRRC8A as critical for cisplatin resistance and suggest LRRC8A-guided patient stratification for ongoing or prospective clinical studies assessing therapy resistance to nanoscale platinum drug nanoformulations versus current standard of care formulations.

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 specific average numbers (2, 6, and 12) of antibodies specific for the dendritic cell (DC) surface receptor, DEC205. We assessed the time-dependent biodistribution of PB-antibody conjugates by in vivo imaging and flow cytometry. We observed that PB-antibody conjugates were trapped in the liver and that the extent of liver accumulation strongly increased with the number of attached antibodies. PB-antibody conjugates were selectively captured in the liver via Fc receptors (FcR) on liver sinusoidal endothelial cells, since systemic administration of FcR-blocking agents or the use of F(ab')2 fragments prevented liver accumulation. Cumulatively, our study demonstrates that liver endothelial cells play a yet scarcely acknowledged role in liver entrapment of antibody-coated NPs and that low antibody numbers on NPs and the use of F(ab')2 antibody fragments are both sufficient for cell type-specific targeting of secondary lymphoid organs and necessary to minimize unwanted liver accumulation.

It remains unclear how the precise length of one-dimensional nanovehicles influences the characters of vaccination. Here, a unimolecular nanovehicle with tailored size and aspect ratio (AR) is applied to deliver CpG oligodeoxynucleotide, a Toll-like receptor (TLR) 9 agonist, as an adjuvant of recombinant hepatitis B virus surface antigen (rHBsAg), for treating chronic hepatitis B (CHB). Cationic nanovehicles with fixed width (ca. 45 nm) but varied length (46 nm-180 nm), AR from 1 to 4, are prepared through controlled polymerization and are loaded with CpG by electrostatic interaction. We reveal that the nanoadjuvant with AR = 2 shows the highest retention in proximal lymph nodes. Importantly, it is more easily internalized into antigen-presenting cells and accumulates in the late endosome, where TLR9 is located. Such a nanoadjuvant exhibits the strongest immune response with rHBsAg to clear the hepatitis B virus in the CHB mouse model, showing that the AR of nanovehicles governs the efficiency of vaccination.

Core cross-linking of polymeric micelles has been demonstrated to contribute to enhanced stability that can improve the therapeutic efficacy. Photochemistry has the potential to provide spatial resolution and on-demand drug release. In this study, light-sensitive polypyridyl-ruthenium(II) complexes were combined with polypept(o)ides for photocleavable core cross-linked polymeric micelles. Block copolymers of polysarcosine-block-poly(glutamic acid) were synthesized by ring-opening N-carboxyanhydride polymerization and modified with aromatic nitrile-groups on the glutamic acid side chain. The modified copolymers self-assembled into micelles and were cross-linked by cis-diaquabis(2,2'-bipyridine)-ruthenium(II) ([Ru(bpy)2(H2O)2]2+) or cis-diaquabis(2,2'-biquinoline)-ruthenium(II) ([Ru(biq)2(H2O)2]2+). Depending on the flexibility and hydrophobicity of the nitrile linker, either small spherical structures (Dh 45 nm, PDI 0.11) or worm-like micelles were obtained. The cross-linking reaction did not affect the overall size distribution but induced a change in the metal-to-ligand charge transfer peak from 482 to 420 nm and 592 to 548 nm. The cross-linked micelles displayed colloidal stability after incubation with human blood plasma and during gel permeation chromatography in hexafluoroisopropanol. Light-induced cleavage of [Ru(bpy)2(H2O)2]2+ was accomplished within 300 s, while [Ru(biq)2(H2O)2]2+ could not be completely released. Analysis in HuH-7 cells revealed increased cytotoxicity via micellar delivery of [Ru(bpy)2(H2O)2]2+ but mostly irradiation damage for [Ru(biq)2(H2O)2]2+. Further evaluation in ovo confirmed stable circulation pointing towards the future development of quick-release complexes.

Radiotherapy of cancer requires both alpha- and beta-particle emitting radionuclides, as these radionuclide types are efficient at destroying different types of tumors. Both classes of radionuclides require a vehicle, such as an antibody or a polymer, to be delivered and retained within the tumor. Polyglutamic acid (pGlu) is a polymer that has proven itself effective as a basis of drug-polymer conjugates in the clinic, while its derivatives have been used for pretargeted tumor imaging in a research setup. trans-Cyclooctene (TCO) modified pGlu is suitable for pretargeted imaging or therapy, as well as for intratumoral radionuclide therapy. In all cases, it becomes indirectly radiolabeled via the bioorthogonal click reaction with the tetrazine (Tz) molecule carrying the radionuclide. In this study, we report the radiolabeling of TCO-modified pGlu with either lutetium-177 (A panel of Tz derivatives containing a metal ion binding chelator (DOTA or macropa) connected to the Tz moiety directly or through a polyethylene glycol (PEG) linker was synthesized and tested for their ability to chelateAll DOTA-modified Tzs efficiently chelatedWe labeled TCO-modified pGlu polymers with alpha- and beta-emitting radionuclides in acceptable RCYs. All

Amphiphilic miktoarm star copolymers have a unique internal structure and many attractive properties with respect to solution self-assembly, such as a high density of internal and peripheral functionalities, low CMC values, and high loading efficiency. However, compared to their linear analogs, the complex architecture demands asymmetric polymer arms emanating from a single core, which poses a significant synthetic challenge. Herein, we demonstrate an approach for the synthesis of polypept(o)ide-based AB3- and A3B-type miktoarm PeptoStars consisting of polypeptidic poly(γ-benzyl-l-glutamate (pGlu(OBn)) as the hydrophobic A arm and polypeptoidic polysarcosine (pSar) as the hydrophilic B arm. These structures, which are completely derived from endogenous amino acids, were realized by the core-first method using a tetrafunctional initiator and the corresponding N-carboxyanhydrides (NCAs) via a controlled, living nucleophilic ring-opening polymerization (ROP). The asymmetric architecture was achieved through an orthogonal protecting group strategy with multiple protection/deprotection steps. Characterization via 1H NMR-, 1H DOSY-spectroscopy, and size-exclusion chromatography (SEC) indicate the presence of well-formed miktoarm PeptoStars with low dispersities (Đ = 1.09–1.14), the precise control over the degree of polymerization and Poisson-like molecular weight distributions. Schwiertz et al. report on the synthesis of miktoarm star polymers based on polypept(o)ides by nucleophilic ring opening polymerization of N-carboxyanhydrides. The reported procedures allow for precise control over chain length, number of arms and end group functionality.

Multidrug-resistant bacterial infections are a global health threat. Nanoparticles are thus investigated as novel antibacterial agents for clinical practice, including wound dressings and implants. We report that nanoparticles' bactericidal activity strongly depends on their physical binding to pathogens, including multidrug-resistant primary clinical isolates, such as Staphylococcus aureus , Klebsiella pneumoniae or Enterococcus faecalis . Using controllable nanoparticle models, we found that nanoparticle-pathogen complex formation was enhanced by small nanoparticle size rather than material or charge, and was prevented by 'stealth' modifications. Nanoparticles seem to preferentially bind to Gram-positive pathogens, such as Listeria monocytogenes, S. aureus or Streptococcus pyrogenes , correlating with enhanced antibacterial activity . Bacterial resistance to metal-based nanoparticles was mediated by biomolecule coronas acquired in pathophysiological environments, such as wounds, the lung, or the blood system. Biomolecule corona formation reduced nanoparticles' binding to pathogens, but did not impact nanoparticle dissolution. Our results provide a mechanistic explanation why nano-sized antibiotics may show reduced activity in clinically relevant environments, and may inspire future nanoantibiotic designs with improved and potentially pathogen-specific activity.

The need for a scalable synthesis of not sequence defined polypeptides as biomaterials is met by the ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCAs). Even though this polymerization technique appears straight forward, it holds pitfalls in terms of reproducibility and overall control over the polymerization conditions, which depends, beside choice of solvent or initiator, significantly on reagent purity. In addition, the synthesis of monomers can lead to the formation of racemic amino acids. Thus, in this work, we describe the benefits of highly pure monomers in order to control nucleophilic ring-opening polymerization NCAs. Hereby, monomer purity is investigated by relating melting points of NCAs with single-crystal and powder X-ray diffraction crystallography data, which further proves retained stereo-information of NCAs.

Iron is an essential co-factor for cellular processes. In the immune system, it can activate macrophages and represents a potential therapeutic for various diseases. To specifically deliver iron to macrophages, iron oxide nanoparticles are embedded in polymeric micelles of reactive polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine). Upon surface functionalization via dihydrolipoic acid, iron oxide cores act as crosslinker themselves and undergo chemoselective disulfide bond formation with the surrounding poly(S-ethylsulfonyl-l-cysteine) block, yielding glutathione-responsive core cross-linked polymeric micelles (CCPMs). When applied to primary murine and human macrophages, these nanoparticles display preferential uptake, sustained intracellular iron release, and induce a strong inflammatory response. This response is also demonstrated in vivo when nanoparticles are intratracheally administered to wild-type C57Bl/6N mice. Most importantly, the controlled release concept to deliver iron oxide in redox-responsive CCPMs induces significantly stronger macrophage activation than any other iron source at identical iron levels (e.g., Feraheme), directing to a new class of immune therapeutics.

Hydrophilic polymers are being investigated as possible coating agents for therapeutic nanoparticles because of their capacity to reduce immune response and increase circulation life time. The mechanism of action of these coatings is not well understood although it is clear that they unspecifically reduce the amount of proteins adsorbing on the nanoparticle surface coming in contact with biological fluids. Here we have investigated, using state-of-the-art atomistic molecular dynamics simulations, the equilibrium and kinetic properties of the interactions forming between human serum albumin, the most abundant protein in the blood stream, and two different and promising polymers poly(ethylene glycol) and poly-sarcosine and we have compared the results with a polymer which is an isomer of poly-sarcosine but has a totally different behavior in terms of adsorption, poly-alanine, because of its well-known aggregation propensity. The results show how the two hydrophilic polymers have a very similar behavior in terms of the amount of polymers adsorbed on the protein surface, pattern of interactions and the kinetics of the adsorption process, with differences emerging due to the different flexibility of the two molecules. In contrast poly-alanine adsorbs significantly more strongly on the protein surface, with a slower kinetics, and a quantitatively, but not qualitatively, different interaction pattern with the surface amino acids with respect to the hydrophilic polymers.

Riboflavin carrier protein (RCP) and riboflavin transporters (RFVTs) have been reported to be highly overexpressed in various cancer cells. Hence, targeting RCP and RFVTs using riboflavin may enhance tumor accumulation and internalization of drug delivery systems. To test this hypothesis, butyl-based 3-arm peptostar polymers were synthesized consisting of a lysine core (10 units per arm) and a sarcosine shell (100 units per arm). The end groups of the arms and the core were successfully modified with riboflavin and the Cy-5.5 fluorescent dye, respectively. While in phosphate buffered saline the functionalized peptostars showed a bimodal behavior and formed supramolecular structures over time, they were stable in the serum maintaining their hydrodynamic diameter of 12 nm. Moreover, the polymers were biocompatible and the uptake of riboflavin targeted peptostars in A431 and PC3 cells was higher than in nontargeted controls and could be blocked competitively. In vivo, the polymers showed a moderate passive tumor accumulation, which was not significantly different between targeted and nontargeted peptostars. Nonetheless, at the histological level, internalization into tumor cells was strongly enhanced for the riboflavin-targeted peptostars. Based on these results, we conclude that passive accumulation is dominating the accumulation of peptostars, while tumor cell internalization is strongly promoted by riboflavin targeting.

Polysarcosine (pSar) is a polypeptoid based on the endogenous amino acid sarcosine (N-methylated glycine), which has previously shown potent stealth properties. Here, lipid nanoparticles (LNPs) for therapeutic application of messenger RNA were assembled using pSarcosinylated lipids as a tool for particle engineering. Using pSar lipids with different polymeric chain lengths and molar fractions enabled the control of the physicochemical characteristics of the LNPs, such as particle size, morphology, and internal structure. In combination with a suited ionizable lipid, LNPs were assembled, which displayed high RNA transfection potency with an improved safety profile after intravenous injection. Notably, a higher protein secretion with a reduced immunostimulatory response was observed when compared to systems based on polyethylene glycol (PEG) lipids. pSarcosinylated nanocarriers showed a lower proinflammatory cytokine secretion and reduced complement activation compared to PEGylated LNPs. In summary, the described pSar-based LNPs enable safe and potent delivery of mRNA, thus signifying an excellent basis for the development of PEG-free RNA therapeutics.

The programming of nanomaterials at molecular length-scales to control architecture and function represents a pinnacle in soft materials synthesis. Although elusive in synthetic materials, Nature has evolutionarily refined macromolecular synthesis with perfect atomic resolution across three-dimensional space that serves specific functions. We show that biomolecules, specifically proteins, provide an intrinsic macromolecular backbone for the construction of anisotropic brush polymers with monodisperse lengths via grafting-from strategy. Using human serum albumin as a model, its sequence was exploited to chemically transform a single cysteine, such that the expression of said functionality is asymmetrically placed along the backbone of the eventual brush polymer. This positional monofunctionalization strategy was connected with biotin-streptavidin interactions to demonstrate the capabilities for site-specific self-assembly to create higher ordered architectures. Supported by systematic experimental and computational studies, we envisioned that this macromolecular platform provides unique avenues and perspectives in macromolecular design for both nanoscience and biomedicine.

The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real-time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse.

The inverse electron demand Diets-Alder (IEDDA) reaction-initiated ligation between 1,2,4,5-tetra-zines (Tz) and trans-cyclooctenes (TCO) is one of the fastest bioorthogonal reactions known today and is therefore increasingly used for in vivo click chemistry. Described herein is the synthesis of Tz- and TCO-functionalised polypeptides, polypeptoids and polypeptide-block-polypeptoids (polypept(o) ides) by ring-opening polymerisation of the corresponding N-carboxyanhydrides using Tz- or TCO-functional amine initiators. Despite the reactivity of tetrazines, polymers with low dispersity and high end group integrity can be obtained as observed by gel permeation chromatography (GPC), nuclear magnetic resonance (NMR) spectroscopy and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Amphiphilic Tz-functionalised block copolypept(o)ides were used to prepare polymeric micelles and organic colloids by miniemulsion techniques, which may find an application as clearing agents in pretargeted nuclear imaging and therapy using efficient in vivo click chemistry. The reaction kinetics of the tetrazine ligation using the synthesised polymers and the accessibility of the Tz groups on the polymeric nanoparticles were evaluated using UV Vis and fluorescence correlation spectroscopy (FCS), and second-order rate constants were determined by stopped-flow spectrophotometry ensuring quantitative conversions in seconds at sub-millimolar concentrations (10-30 s).

Homocysteine and cysteine are the only natural occurring amino acids that are capable of disulfide bond formations in peptides and proteins. The chemoselective formation of asymmetric disulfide bonds, however, is chemically challenging and requires an activating group combining stability against hard nucleophiles, e.g., amines, with reactivity toward thiols and soft nucleophiles. In light of these considerations, we introduced the S-alkylsulfonyl cysteines in our previous work. Here, we present the synthesis and ring-opening polymerization of S-ethylsulfonyl-l-homocysteine N-carboxyanhydrides. We demonstrate that the polymerization leads to narrowly distributed polypeptides (Đ = 1.1–1.3) with no detectable side reactions in a chain length regime from 11 to 165. In contrast to the already reported cysteine derivatives, poly(S-ethylsulfonyl-l-homocysteine)s do not form β-sheets, which reduce solubility and limit the degree of polymerization of poly(S-ethylsulfonyl-l-cysteine)s to 50. Instead, these polymers...

Circulation lifetime is a crucial parameter for a successful therapy with nanoparticles. Reduction and alteration of opsonization profiles by surface modification of nanoparticles is the main strategy to achieve this objective. In clinical settings, PEGylation is the most relevant strategy to enhance blood circulation, yet it has drawbacks, including hypersensitivity reactions in some patients treated with PEGylated nanoparticles, which fuel the search for alternative strategies. In this work, lipopolysarcosine derivatives (BA-pSar, bisalkyl polysarcosine) with precise chain lengths and low polydispersity indices are synthesized, characterized, and incorporated into the bilayer of preformed liposomes via a post insertion technique. Successful incorporation of BA-pSar can be realized in a clinically relevant liposomal formulation. Furthermore, BA-pSar provides excellent surface charge shielding potential for charged liposomes and renders their surface neutral. Pharmacokinetic investigations in a zebrafish model show enhanced circulation properties and reduction in macrophage recognition, matching the behavior of PEGylated liposomes. Moreover, complement activation, which is a key factor in hypersensitivity reactions caused by PEGylated liposomes, can be reduced by modifying the surface of liposomes with an acetylated BA-pSar derivative. Hence, this study presents an alternative surface modification strategy with similar benefits as the established PEGylation of nanoparticles, but with the potential of reducing its drawbacks.

The use of PEGylated lipids for the synthesis of stealth liposomes and lipid formulations for nucleic acid delivery has promoted the development of nanoparticle based drugs for cancer therapy, and chronic diseases. Moreover, several other nanomedicines based on these materials have advanced into clinical trails. This enormous success, however, has recently been compromised by the occurrence of immune responses towards PEG, which render pharmacokinetics and can substantially reduce the therapeutic efficiency of drugs. Therefore, alternatives for PEGylated lipids with comparable or even identical solution properties are required. In this work, we report the synthesis of polysarcosine based lipopolymers, which combine the stealth material polysarcosine with bisalkyl-amines. The lipopolymers are obtained by nucleophilic ring opening polymerization of sarcosine NCAs using C-12 and C-18 based bisalkyl amines, namely, didodecyl and dioctadecyl amine. To achieve a controlled living polymerization conditions have been optimized yielding the desired lipopolymers with precise control over chain length, end group integrity and low polymer dispersity (D < 1.2) as demonstrated by size exclusion chromatography, H-1 DOSY nuclear magnetic resonance spectroscopy and MALDI-ToF mass spectroscopy. In addition, the critical micelle concentrations and the incorporation of these lipids in lipid bilayers are reported. Therefore, this work sets the synthetic foundation for the development of alternatives to PEGylated lipids.

Prevention of biofouling and microbial contamination of implanted biomedical devices is essential to maintain their functionality and biocompatibility. For this purpose, polypept(o)ide block copolymers have been developed, in which a protein-resistant polysarcosine (pSar) block is combined with a dopamine-modified poly(glutamic acid) block for surface coating and silver nanoparticles (Ag NPs) formation. In the development of a novel, versatile, and biocompatible antibacterial surface coating, block lengths pSar were varied to derive structure-property relationships. Notably, the catechol moiety performs two important tasks in parallel; primarily it acts as an efficient anchoring group to metal oxide surfaces, while it furthermore induces the formation of Ag NPs. Attributing to the dual function of catechol moieties, antifouling pSar brush and antimicrobial Ag NPs can not only adhere stably on metal oxide surfaces, but also display passive antifouling and active antimicrobial activity, showing good biocompatibility simultaneously. The developed strategy seems to provide a promising platform for functional modification of biomaterials surface to preserve their performance while reducing the risk of bacterial infections.

Functionalization of macromolecules (antibodies, polymers, nanoparticles) with click-reactive groups greatly enhances the versatility of their potential applications. Click chemistry based on tetrazine - trans-cyclooctene (TCO) ligation is especially promising and is already widely applied for pretargeted imaging and therapy. Indirect radiolabeling of TCO-functionalized macromolecules with substoichiometric amounts of radioactive tetrazines is a convenient way to monitor the fate of those macromolecules by means of positron emission tomography (PET) imaging after their administration into the test subject. In this work, the preparation is reported of TCO-containing graft copolymers, namely PeptoBrushes (polyglutamic acid-graft-polysarcosine), novel [11C]carboxylated tetrazines, and their combined use in radiolabeling the polymer by inverse electron demand Diels Alder reaction, to investigate it is potential for an application in pretarget imaging or injectable brachytherapy. The procedure for [11C]tetrazine production is easy and scalable, while indirect TCO-PeptoBrushes labeling with these [11C]tetrazines is mild, fast, and quantitative. This strategy allows facile 11C-labeling of diverse TCO-functionalized macromolecules, so that their localization and distribution shortly after injection can be assessed by PET.

Immunotherapies have the potential to significantly advance treatment of inflammatory disease and cancer, which are in large part driven by immune cells. Selectins control the first step in immune cell adhesion and extravasation, thereby guiding leukocyte trafficking to tissue lesions. We analyzed four different highly specific selectin-binding glycopolymers, based on linear poly(2-hydroxypropyl)-methacrylamide (PHPMA) polymers. These glycopolymers contain either the tetrasaccharide sialyl-LewisX (SLeX) or the individual carbohydrates fucose, galactose, and sialic acids mimicking the complex SLeX binding motive. The glycopolymers strongly bind to primary human macrophages, without activating them, and also to primary human blood leukocytes, but poorly to fibroblasts and endothelial cells in vitro. After intravenous injection in mice, all glycopolymers accumulated in the liver without causing hepatotoxicity. The glycosylated binder most potently targeted resident hepatic macrophages (Kupffer cells) and pro...

Prazise Kontrolle uber Morphologie und Funktion polymerer Nanostrukturen im Rahmen der Selbstorganisation stellt nach wie vor eine Herausforderung im Feld der Material- und biomedizinischen Wissenschaften dar, insbesondere wenn unabhangige Kontrolle uber einzelne Partikeleigenschaften erwunscht ist. Hier wird uber Sekundarstruktur-gesteuerte Selbstorganisation von Nanostrukturen basierend auf amphiphilen Blockcopolypept(o)iden berichtet und eine Strategie zur bio-reversiblen Einstellung der Kernpolaritat und –funktion unabhangig von der Partikelpraparation vorgestellt. Der Peptiden eigene Prozess der Sekundarstrukturbildung erlaubt so die Herstellung spharischer und wurmartiger kernvernetzter Architekturen ausgehend von ein und demselben Blockcopolymer und bietet einen simplen, aber dennoch leistungsfahigen Ansatz zur Herstellung vielseitiger peptidbasierter Nanopartikel.

Functional amino acids whose reactivity is compatible with the polymerization of α-amino acid-N-carboxyanhydrides (NCAs) have received a lot of attention in recent years. The appeal of these reactive monomers lies in the fact that the resulting polymers can be easily modified in one controlled post-polymerization step, leading to a variety of polypeptidic materials like helical non-natural polycations or glycopeptides. This review highlights recent developments in the field and focuses on the different reactive groups like alkynes, alkenes, azides, chlorides and S-alkylsulfonyls. Furthermore, the modifications after polymerization are discussed, pointing out advantages and challenges. Besides orthogonal functionalities in the side chain, different approaches are summarized to modify α- and ω-chain ends with orthogonal functionalities for grafting to and grafting from applications. Thus, new materials can be produced through mild modifications as presented. Finally, we also highlight the development of orthogonally reactive NCAs for the synthesis of polypeptoids, a field that is relatively unexplored, but offers great possibilities for example for polypept(o)idic hybrid materials.