Polypeptide Nanoparticles Research Articles

Diradicaloid-Loaded Polypeptide Nanoparticles for Two-Photon NIR Phototheranostics.

Stable organic radicals, with unique electronic transitions from the ground state (D0) to the doublet excited state (D1), show promise as high-fluorescence quantum yield dyes. While organic small-molecule photosensitizers (PSs) have advanced for tumor photodynamic therapy (PDT), opportunities exist to enhance their performance and functionality. Herein, we synthesized Thiele's fluorocarbon derivative diradicaloid TFC-I with nearly 100% PLQY and integrated it into amphiphilic polypeptide nanoparticles, P-TI, using a precursor-doping approach. P-TI demonstrated notable features including high photostability, aggregation-induced emission, bright near-infrared fluorescence, substantial quantum yield (37% PLQY), robust near-infrared two-photon absorption (∼400 GM cross section), and superior ROS generation compared to commercial PSs. In vitro and in vivo experiments confirmed that P-TI performed well in mitochondria-targeted PDT, two-photon fluorescence imaging, and biosafety. This work highlights the use of organic stable radicals with precursor-doping for efficient PDT and deep tumor tissue imaging.

Tumor microenvironment-activated polypeptide nanoparticles for oncolytic immunotherapy

Cationic oncolytic polypeptides have gained increasing attention owing to their ability to directly lyse cancer cells and activate potent antitumor immunity. However, the low tumor cell selectivity and inherent toxicity induced by positive charges of oncolytic polypeptides hinder their systemic application. Herein, a tumor microenvironment-responsive nanoparticle (DNP) is developed by the self-assembly of a cationic oncolytic polypeptide (PLP) with a pH-sensitive anionic polypeptide via electrostatic interactions. After the formation of DNP, the positive charges of PLP are shielded. DNPs can keep stable in physiological conditions (pH 7.4) but respond to acidic tumor microenvironment (pH 6.8) to release oncolytic PLP. As a result, DNPs evoke potent immunogenic cell death by disrupting cell membranes, damaging mitochondria and increasing intracellular levels of reactive oxygen species. In vivo results indicate that DNPs significantly improve the biocompatibility of PLP, and inhibit tumor growth, recurrence and metastasis by direct oncolysis and activation of antitumor immune responses. In summary, these results indicate that pH-sensitive DNPs represent a prospective strategy to improve the tumor selectivity and biosafety of cationic polymers for oncolytic immunotherapy.

Novel injectable polypeptide nanoparticle encapsulated siRNA targeting TGF-β1 and COX-2 for localized fat reduction I: Preclinical invitro and animal models.

Obesity and localized fat accumulation continue to drive the demand for minimally invasive body contouring technologies including injectable compounds for local fat reduction. siRNA offers a potential for an injectable to specifically target and silence genes involved in adipogenesis with minimal inflammatory side effects. This study evaluates the efficacy of STP705, an injectable containing siRNA encapsulated within histidine-lysine polypeptide (HKP) nanoparticles targeting transforming growth factor β1 (TGF-β1) and cyclooxygenase-2 (COX-2), crucial mediators in adipocyte differentiation and fat retention, using invitro, porcine, and murine models. In vitro experiments on mouse preadipocytes and invivo trials using Diet Induced Obese (DIO) mice and Yucatan minipigs were conducted to assess the gene silencing efficiency, tissue localization, pharmacodynamics, and safety profile of STP705. STP705 effectively reduced the expression of TGF-β1 and COX-2, with a notable decrease in adipocyte volume and lipid content without adverse systemic effects. In DIO mice, the HKP-siRNA complex demonstrated precise localization to injected adipose tissue, maintaining significant gene silencing, and detectable levels of siRNA for up to 14 days post-administration. Similar results in minipigs showed a significant reduction in subcutaneous adipose tissue thickness. The results of these studies support the use of targeted siRNA therapy specifically targeting TGF-β1 and COX-2, for localized fat reduction, offering a potential minimally invasive alternative to current fat reduction methods.

Synthesis of D-A-type groups modified aza-BODIPY fluorescent dye encapsulated by amphiphilic polypeptide nanoparticles for NIR-II phototheranostics

An innovative organic small molecule with a D-A structure was synthesized by connecting triphenylamine to BODIPY via a thiophene bridge. Triphenylamine and thiophene units ingeniously modulate the balance between steric hindrance and π-π interactions around the flat aza-BODIPY core. The molecule exhibits near-infrared fluorescence absorption and emits at roughly 1100 nm, featuring a significant Stokes shift. Both the molecule and its nanoparticles demonstrate high stability and achieve a remarkable 35 % photothermal conversion efficiency when conjugated with the P(OEGMA)20-P(Asp)14 copolymer. In vitro assessments show low dark toxicity and outstanding biocompatibility. Moreover, in vivo studies and photothermal therapy in mice indicate substantial tumor shrinkage and reduced recurrence, confirming its potential in cancer treatment. These results highlight the promise of this organic molecule and its nanoparticles for NIR-II imaging-guided photothermal therapy, introducing a novel approach to phototheranostic applications for cancer management.

Polypeptide nanoparticles conjugated with an NIR-II organic dye for TRPV1 channel blockade enhance mild phototheranostics

Photothermal therapy (PTT) has attracted attention as a highly effective non-invasive treatment method. However, the high localized temperatures (>50 °C) required for its treatment will inevitably cause damage to the surrounding normal tissues. Therefore, it is important to develop novel and effective strategies to achieve mild photothermal therapy (mPTT). The overexpression of heat shock proteins (HSPs), a widespread heat stress protein, leads to the generation of heat resistance in cancer cells, which seriously affects the therapeutic effect. Thus, inhibiting the expression of HSPs to reduce the heat resistance of tumor cells is expected to enhance the therapeutic effect of mPTT. Here, we successfully synthesized a fluorescent probe bonded with an amphiphilic polypeptide to a cyanine dye and achieved physical encapsulation of the blocker SB705498 through a self-assembly process. SB705498 promotes transient receptor potential vanilloid member 1 (TRPV1) channel blockade that can inhibit the translocation of the heat shock transcription factor 1 (HSF 1) by blocking the influx of calcium and thus affecting the expression of HSPs, which has the potential to enhance the thermotherapy of cancer under mild conditions. In addition, the nanoparticles enabled NIR-II fluorescence imaging with good stability and high photothermal conversion efficiency (48.10 %). Therefore, this study provides a new strategy for realizing precise mPTT(<45 °C) guided by NIR-II imaging. Statement of significanceInhibition of overexpression of heat shock proteins (HSPs) in cancer photothermal therapy (PTT) is expected to enhance the therapeutic effect of mild photothermal therapy (mPTT). In this study, we synthesized a fluorescent probe bonded to cyanine dyes with amphiphilic polypeptides and physically wrapped the blocker SB705498 through a self-assembly process. As a transient receptor potential vanillin 1 (TRPV1) channel blocker, SB705498 inhibits heat shock transcription factor 1 (HSF1) translocation by blocking calcium ion influx, thereby improving mPTT efficacy by inhibiting the expression of HSPs. The nanoparticles also enable NIR-II fluorescence imaging with good stability and high photothermal conversion efficiency (48.10 %). Thus, this study provides a new strategy for NIR-II mPTT.

ATP Inhibition for Starvation/Mild Photothermal/Photodynamic Synergy Therapy Using Polypeptide Nanoparticles Conjugating 2-Deoxy-D-Glucose and Dye under NIR Phototheranostic Strategy.

Rapid propagation of tumor cells requires plenty of energy, which is adenosine triphosphate (ATP) dependent. ATP inhibition in tumors not only results in the starvation of tumor cells but also down-regulation of the level of heat shock proteins (HSPs), which usually increase during traditional photothermal therapy (PTT), especially when the temperature is up 50°C. 2-deoxy-D-glucose (2DG) is an anti-glycolytic reagent and can be used as an efficient agent for ATP inhibition in tumors. Compared with typical PTT, low-temperature mild photothermal therapy (MPTT) is receiving more and more attention because it avoids the high temperatures causing damage to the normal tissue, and the increase of HSPs which decrease PTT. Here, multifunctional polypeptide nanoparticles pDG@Ahx conjugating both a NIR probe Ahx-BDP and 2DG into the side chain of the amphiphilic polypeptide have been prepared. In vitro and in vivo studies reveal that the as-prepared nanoparticles achieve a synergistic effect of starvation/MPTT/PDT (photodynamic therapy), and it provides a new strategy to NIR-I/II fluorescence imaging-guided starvation/MPTT/PDT synergy therapy for tumors.

Self-enhanced regulation of stable organic radicals with polypeptide nanoparticles for mild second near-infrared phototheranostics

Stable organic radicals have emerged as a promising option to enhance fluorescence quantum yield (QY), gaining traction in medical treatment due to their unique electronic transitions from the ground state (D0) to the doublet excited state (D1). We synthesized a stable dicyanomethyl radical with a NIR-II fluorescence QY of 0.86 %, surpassing many NIR-II organic dyes. Subsequently, amphiphilic polymer-encapsulated nanoparticles (NPs) containing the radical were created, achieving a NIR-II fluorescence QY of 0.32 %, facilitating high-contrast bio-imaging. These CNPPs exhibit self-enhanced photothermal properties, elevating photothermal conversion efficiency (PCE) from 43.5 % to 57.5 % under 915 nm laser irradiation. This advancement enables more efficient photothermal therapy (PTT) with lower dye concentrations and reduced laser power, enhancing both feasibility and safety. Through regular fractionated mild photothermal therapy, we observed the release of damage-associated molecular patterns (DAMPs) and an increase in cytokine expression, culminating in combined mild phototherapy (m-PTT)-mediated immunogenic cell death (ICD). Consequently, we developed an immunostimulatory tumor vaccine, showcasing a novel approach for refining photothermal agents (PTA) and optimizing the PTT process.

Optimization of Dendritic Polypeptide Delivery System for Antisense Antibacterial Agents Targeting ftsZ.

There is an urgent requirement for a novel treatment strategy for drug-resistant Staphylococcus aureus (S. aureus) infection. Antisense antimicrobials are promising antimicrobials, and efficient drug delivery systems are necessary for the further development of antisense antimicrobials. To develop new antisense drugs and further improve delivery efficiency and safety, we designed and screened new antisense sequences and optimized dendritic polypeptide nanoparticles (DP-AD) discovered in previous studies. The N/P ratio is optimized from 8:1 to 6:1, and the positive charge number of the optimized DP-AD is studied comprehensively. The results show that the N/P ratio and positive charge number have no significant effect on the particle size distribution and transport efficiency of DP-AD. Reducing the N/P ratio can significantly reduce the cytotoxicity of DP-AD, but it does not affect its delivery efficiency and antibacterial activity. However, in drug-resistant strains, the antibacterial activity of DP-AD76:1 with 10 positive charges is higher than that of DP-AD86:1 with 8 positive charges. Our research discovered a novel ASOs targeting ftsZ and concluded that DP-AD76:1 with 10 positive charges was the optimal choice at the current stage, which provided a promising strategy for the treatment of drug-resistant S. aureus.

Codelivery of TGFβ and Cox2 siRNA inhibits HCC by promoting T-cell penetration into the tumor and improves response to Immune Checkpoint Inhibitors.

Upregulation of TGFβ and Cox2 in the tumor microenvironment results in blockade of T-cell penetration into the tumor. Without access to tumor antigens, the T-cell response will not benefit from administration of the immune checkpoint antibodies. We created an intravenous polypeptide nanoparticle that can deliver two siRNAs (silencing TGFβ and Cox2). Systemic administration in mice, bearing a syngeneic orthotopic hepatocellular carcinoma (HCC), delivers the siRNAs to various cells in the liver, and significantly reduces the tumor. At 2mg/kg (BIW) the nanoparticle demonstrated a single agent action and induced tumor growth inhibition to undetectable levels after five doses. Reducing the siRNAs to 1mg/kg BIW demonstrated greater inhibition in the presence of PD-L1 mAbs. After only three doses BIW, we could still recover a smaller tumor and, in tumor sections, showed an increase in penetration of CD4+and CD8+T-cells deeper into the remaining tumor that was not evident in animals treated with non-silencing siRNA. The combination of TGFβ and Cox2 siRNA co-administered in a polypeptide nanoparticle can act as a novel therapeutic alone against HCC and may augment the activity of the immune checkpoint antibodies. Silencing TGFβ and Cox2 converts an immune excluded (cold) tumor into a T-cell inflamed (hot) tumor.

Doxorubicin delivery systems based on polypeptide nanoparticles for subcutaneous administration in cancer therapy

Polypeptide-based doxorubicin delivery systems were prepared and their physicochemical and functional properties, such as size, encapsulation efficiency, stability, release of doxorubicin in various media and cytotoxicity, were characterized. An in vivo study revealed an effective reduction of tumor growth when these systems were administered subcutaneously.

Fabrication of Oleophilic Polypeptide Nanoparticle from Complexing of Cross-Linked Epsilon-poly-l-lysine with Docusate Sodium for Preparation of Bactericidal Thermoplastic Polyurethanes.

Thermoplastic polyurethanes (TPUs) are extensively utilized in the biomedical field due to their exceptional mechanical properties and biocompatibility. However, the lack of antibacterial activity limits their application ranges. Nanoscopic particle-based additives with inherent antibacterial characteristics are regarded as promising strategies to prevent biomaterials-associated infection. Herein, a novel polymeric nanoparticle is prepared, which integrates chemically cross-linked epsilon-poly-l-lysine (CPL) and anionic surfactant-docusate sodium (DS). The cross-linked epsilon-poly-l-lysine/docusate sodium (CPL/DS) nanoparticle can be well dispersed in organic solvent and a polymer matrix, which is beneficial to endowing TPUs with synergistic miscibility and antibacterial properties. An antibacterial test showed that the CPL/DS nanoparticles have strong antibacterial activity against S. aureus. Moreover, the results of antibacterial experiments in vitro revealed that almost 100% of S. aureus could be killed by CPL/DS nanoparticle-embedded TPU film with a content of 0.5 wt %. In addition, all of the CPL/DS modified TPU films showed good cytocompatibility in vitro. Consequently, this kind of CPL/DS nanoplatform has great potential to serve as a safe and high-efficient bactericidal agent for endowing biomedical devices with bactericidal property.

Disease-specific corona mediated co-delivery of MTX and siRNA-TNFα by a polypeptide nanoplatform with antigen-scavenging functions in psoriasis

Psoriatic self-antigen LL37 has the ability to stimulate skin-homing T lymphocytes, which is the constitutive factor for the relapse of psoriasis. The local inflammatory modulation in psoriatic skin is not sufficient for the restoration of immune homeostasis. In this study, we found the expression levels of disease specific proteins including LL37, S100A8 and S100A9 were significantly increased in psoriasis. The self-assembled nanoparticles based on methoxy-poly(ethylene glycol)-block-poly(L-histidine) and methoxy-poly(ethylene glycol)-block-poly(L-lactic acid) (HLNPs) were capable of interacting and absorbing these disease specific proteins, leading to the accumulation of HLNPs in peripheral inflammatory immune cells. Therefore, we proposed an “Antigen-scavenging” polypeptide nano-system assembled from the “sandwich” structure composed of HLNPs, siRNA-TNFα, and oligolysine conjugated methotrexate prodrug (HLNP-MNs), with the encapsulation of ROS-responsive methotrexate prodrug and siRNA-TNFα. We evaluated the biological fate of HLNP-MNs in vivo, the ROS-responsive release of methotrexate (MTX) and siRNA-TNFα in inflammatory immune cells, the suppression of immune response triggered by autoantigen LL37 and the inhibition of skin-homing T lymphocytes from the aspects of “nanoparticle-protein” interaction, cellular level and mouse models. The design of “Antigen-scavenging” polypeptide nanoparticles with loading of MTX and siRNA-TNFα, can shed light on restoring immune homeostasis in psoriasis.

Carfilzomib-Loaded Ternary Polypeptide Nanoparticles Stabilized by Polycationic Complexation

Carfilzomib (CFZ) is a second-generation proteasome inhibitor showing great efficacy in multiple myeloma treatment, yet its clinical applications for other diseases such as solid cancers are limited due to low aqueous solubility and poor biostability. Ternary polypeptide nanoparticles (tPNPs) are drug carriers that we previously reported to overcome these pharmaceutical limitations by entrapping CFZ in the core of the nanoparticles and protecting the drugs from degradation in biological media. However, preclinical studies revealed that tPNPs would require further improvement in particle stability to suppress initial burst drug release and thus achieve prolonged inhibition of proteasome activity with CFZ against tumor cells in vivo. In this study, CFZ-loaded tPNPs are stabilized by polycations which have varying pKa values and thus differently modulate nanoparticle stability in response to solution pH. Through polyion complexation, the polycations appeared to stabilize the core of tPNPs entrapping CFZ-cyclodextrin inclusion complexes while allowing for uniform particle size before and after freeze drying. Interestingly, CFZ-loaded tPNPs (CFZ/tPNPs) showed pH-dependent drug release kinetics, which accelerated CFZ release as solution acidity increased (pH < 6) without compromising particle stability at the physiological condition (pH 7.4). In vitro cytotoxicity and proteasome activity assays confirmed that tPNPs stabilized with cationic polymers improved bioactivity of CFZ against CFZ-resistant cancer cells, which would be greatly beneficial in combination with pH-dependent drug release for treatment of solid cancers with drug resistance and tumor microenvironment acidosis by using CFZ and other proteasome inhibitors.

A Dendrimer Peptide (KK2DP7) Delivery System with Dual Functions of Lymph Node Targeting and Immune Adjuvants as a General Strategy for Cancer Immunotherapy

The clinical efficacy of personalized cancer vaccines still needs to be improved due to their insufficient immune effect. The development of innovative adjuvants and lymph node-targeted delivery systems is the key to improving the clinical efficacy of personalized vaccines. However, there is still a lack of an adjuvant delivery system that is simple in preparation and capable of mass production and integrates adjuvant and lymph node targeted delivery functions. Here, this work reports that a simple dendrimer polypeptide (KK2DP7) nanoparticle enhances the immune efficacy of an OVA/neoantigen-based vaccine. Due to its multiple functions as a delivery vehicle, immune adjuvant, and facilitator of dendritic cell migration, KK2DP7 efficiently increases the efficiency of antigen uptake and cross-presentation by antigen-presenting cells (APCs) and delivers antigens to lymph nodes via APCs. Strikingly, the antitumor effect of KK2DP7/OVA is superior to that of commonly used adjuvants such as poly(I:C), CpG, and aluminum adjuvant combined with OVA. Furthermore, KK2DP7/OVA combined with anti-PD-1 antibody is able to prevent tumor recurrence in a postoperative recurrent tumor model. Thus, KK2DP7-based cancer vaccines alone or in combination with immune checkpoint blockade therapies to treat tumors or postoperative tumor recurrence are a powerful strategy to enhance antitumor immunity.

Targeting Human Endothelial Cells with Glutathione and Alanine Increases the Crossing of a Polypeptide Nanocarrier through a Blood-Brain Barrier Model and Entry to Human Brain Organoids.

Nanoparticles (NPs) are the focus of research efforts that aim to develop successful drug delivery systems for the brain. Polypeptide nanocarriers are versatile platforms and combine high functionality with good biocompatibility and biodegradability. The key to the efficient brain delivery of NPs is the specific targeting of cerebral endothelial cells that form the blood-brain barrier (BBB). We have previously discovered that the combination of two different ligands of BBB nutrient transporters, alanine and glutathione, increases the permeability of vesicular NPs across the BBB. Our aim here was to investigate whether the combination of these molecules can also promote the efficient transfer of 3-armed poly(l-glutamic acid) NPs across a human endothelial cell and brain pericyte BBB co-culture model. Alanine and glutathione dual-targeted polypeptide NPs showed good cytocompatibility and elevated cellular uptake in a time-dependent and active manner. Targeted NPs had a higher permeability across the BBB model and could subsequently enter midbrain-like organoids derived from healthy and Parkinson's disease patient-specific stem cells. These results indicate that poly(l-glutamic acid) NPs can be used as nanocarriers for nervous system application and that the right combination of molecules that target cerebral endothelial cells, in this case alanine and glutathione, can facilitate drug delivery to the brain.

Chiral polypeptide nanoparticles as nanoadjuvants of nanovaccines for efficient cancer prevention and therapy

The chirality of bioactive molecules is closely related to their functions. D-amino acids commonly distributed in the bacterial cell walls trigger a robust anti-infective immune response. Inspired by that, two kinds of chiral polypeptides, poly(L-phenylalanine)-block-poly(L-lysine) (PL-K) and poly(L-phenylalanine)-block-poly(D-lysine) (PD-K), were synthesized and used as nanoadjuvants of nanovaccines for cancer prevention and therapy. The amphiphilic polypeptides self-assembled into nanoparticles with a diameter of about 30 nm during ultrasonic-assisted dissolution in phosphate-buffered saline. The nanovaccines PL-K-OVA and PD-K-OVA were easily prepared by mixing solutions of PL-K or PD-K and the model antigen chicken ovalbumin (OVA), respectively, with loading efficiencies of almost 100%. Compared to PL-K-OVA, PD-K-OVA more robustly induced dendritic cell maturation, antigen cross-presentation, and adaptive immune response. More importantly, it effectively prevented and treated the OVA-expressed B16-OVA melanoma model. PD-K-OVA achieved a tumor inhibition rate of 94.9% and even 97.0% by combining with anti-PD-1 antibody. Therefore, the chiral polypeptide nanoparticles represent simple, efficient, and extensively applicable nanoadjuvants for various nanovaccines.

IL-2-loaded Polypeptide Nanoparticles for Enhanced Anti-cancer Immunotherapy

IL-2-loaded Polypeptide Nanoparticles for Enhanced Anti-cancer Immunotherapy

Synthesis of pH-responsive supramolecular polypeptide nanoparticles from α-amino acids for combined chemo-photothermal therapy

A new smart supramolecular polypeptide copolymer P(Glu-co-Lys) was synthesized by the polymerization of α-amino acids using the N-thiocarboxylic acid anhydride (NTA) method, using the pH dynamic response peptide of L-glutamic acid and L-lysine as a carrier for tumor cells. The drug delivery system activated by external acid can self-assemble (pH 7.4) and disassemble (pH 5.5) under the adjustment of pH to load the drug and control its release. Doxycycline (DOX) and the photothermal reagent hydrophilic quanternary stereo-cyanine (HQS-Cy) were loaded into the peptide copolymer to obtain HQS-Cy/DOX nanoparticles (NPs) for chemo-photothermal therapy. Gentle photothermal heating can enhance the absorption of drugs by cells and enhance the efficacy of chemotherapy. In addition, chemo-photothermal therapy can solve the defect of easy recurrence after single photothermal therapy. The ingenious nanodrug delivery system of HQS-Cy/DOX NPs provides great potential for the improvement of chemo-photothermal therapy and will achieve excellent therapeutic effects in cancer treatment.

Stimuli-Responsive Polypeptide Nanoparticles for Enhanced DNA Delivery

The development of non-viral delivery systems for effective gene therapy is one of the current challenges in modern biomedicinal chemistry. In this paper, the synthesis of pH- and redox-responsive amphiphilic polypeptides for intracellular DNA delivery is reported and discussed. Two series of polypeptides consisting of L-lysine, L-phenylalanine, L-histidine, and L-cysteine as well as the same amino acids with L-glutamic acid were synthesized by a combination of copolymerization of N-carboxyanhydrides of α-amino acids and post-polymerization modification of the resulting copolymers. The presence of histidine provided pH-sensitive properties under weakly acidic conditions specific to endosomal pH. In turn, the presence of cysteine allowed for the formation of redox-responsive disulfide bonds, which stabilized the self-assembled nanoparticles in the extracellular environment but could degrade inside the cell. The formation of intraparticle disulfide bonds resulted in their compactization from 200-250 to 55-100 nm. Empty and pDNA-loaded cross-linked nanoparticles showed enhanced stability in various media compared to non-crosslinked nanoparticles. At the same time, the addition of glutathione promoted particle degradation and nucleic acid release. The delivery systems were able to retain their size and surface charge at polypeptide/pDNA ratios of 10 or higher. GFP expression in HEK 293 was induced by the delivery of pEGFP-N3 with the developed polypeptide nanoparticles. The maximal transfection efficacy (70%) was observed when the polypeptide/pDNA ratio was 100.

Betv1-displaying elastin-like polypeptide nanoparticles induce a strong humoral and weak CD4+ T-cell response against Betv1 in a murine immunogenicity model.

There is growing concern about the toxicity of colloidal aluminum salts used as adjuvants in subcutaneous allergen immunotherapy (SCIT). Therefore, alternative adjuvants and delivery systems are being explored to replace alum in SCIT. We applied micellar elastin-like polypeptides (ELPs), a type of self-assembling protein, to replace alum as vaccine adjuvant in birch pollen SCIT. ELP and an ELP-Bet v 1 fusion protein were expressed in E. coli and purified by immuno-affinity chromatography and inverse-transition cycling (ITC). Nanoparticles self-assembled from ELP and a 9:1 ELP/ELP-Bet v 1 mixture were characterized by using dynamic light scattering and atomic force microscopy. Allergenicity was assessed by measuring mediator release from rat basophilic leukemia cells transformed with the human FcϵR1 and sensitized with sera derived from human birch pollen allergic patients. Humoral and T-cell immunity were investigated by immunizing naïve mice with the ELP/ELP-Bet v 1 nanoparticles or alum-adsorbed Bet v 1, both containing 36 µg Bet v 1. ELP and ELP/ELP-Bet v 1 self-assembled at 37°C into spherically shaped micelles with a diameter of ~45 nm. ELP conjugation made Bet v 1 hypo-allergenic (10-fold). Compared to alum-adsorbed Bet v 1, ELP/ELP-Bet v 1 nanoparticles induced stronger IgG responses with an earlier onset. Additionally, ELP/ELP-Bet v 1 did not induce Th2 skewing cytokines and IgE. The hypoallergenic character and strong humoral immune response in the absence of a Th2-skewing T-cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT.