Synthetic molecular motor activates drug delivery from polymersomes.

The design of stimuli-responsive systems in nanomedicine arises from the challenges associated with the unsolved needs of current molecular drug delivery. Here, we present a delivery system with high spatiotemporal control and tunable release profiles. The design is based on the combination of an hydrophobic synthetic molecular rotary motor and a PDMS-b-PMOXA diblock copolymer to create a responsive self-assembled system. The successful incorporation and selective activation by low-power visible light (λ = 430 nm, 6.9 mW) allowed to trigger the delivery of a fluorescent dye with high efficiencies (up to 75%). Moreover, we proved the ability to turn on and off the responsive behavior on demand over sequential cycles. Low concentrations of photoresponsive units (down to 1 mol% of molecular motor) are shown to effectively promote release. Our system was also tested under relevant physiological conditions using a lung cancer cell line and the encapsulation of an Food and Drug Administration (FDA)-approved drug. Similar levels of cell viability are observed compared to the free given drug showing the potential of our platform to deliver functional drugs on request with high efficiency. This work provides an important step for the application of synthetic molecular machines in the next generation of smart delivery systems.

Glutathione-Responsive Methotrexate Polymersomes for Potential Management of Ectopic Pregnancy.

The first-line treatment for ectopic pregnancy (EP), the chemotherapeutic methotrexate (MTX), has a failure rate of more than 10%, which can lead to severe complications or death. Inadequate accumulation of administered MTX at the ectopic implantation site significantly contributes to therapeutic failure. This study reports the first glutathione-responsive polymersomes for efficient delivery of MTX to the implantation site and its triggered release in placental cells. Fluorescence and photoacoustic imaging have confirmed that the developed polymersomes preferentially accumulate after systemic administration in the implantation site of pregnant mice at early gestational stages. The high concentrations of intracellular glutathione (GSH) reduce an incorporated disulfide bond within polymersomes upon internalization into placental cells, resulting in their disintegration and efficient drug release. Consequently, MTX delivered by polymersomes induces pregnancy demise in mice, as opposed to free MTX at the same dose regimen. To achieve the same therapeutic efficacy with free MTX, a sixfold increase in dosage is required. In addition, mice successfully conceive and birth healthy pups following a prior complete pregnancy demise induced by methotrexate polymersomes. Therefore, the developed MTX nanomedicine can potentially improve EP management and reduce associated mortality rates and related cost.

Constructing artificial respiratory chain in polymer compartments: Insights into the interplay between bo3 oxidase and the membrane.

Cytochrome bo3 ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F1Fo-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo3 oxidase in giant unilamellar vesicles made of PDMS-g-PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min-1 To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS-g-PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.

Organelle-specific targeting of polymersomes into the cell nucleus.

Organelle-specific nanocarriers (NCs) are highly sought after for delivering therapeutic agents into the cell nucleus. This necessitates nucleocytoplasmic transport (NCT) to bypass nuclear pore complexes (NPCs). However, little is known as to how comparably large NCs infiltrate this vital intracellular barrier to enter the nuclear interior. Here, we developed nuclear localization signal (NLS)-conjugated polymersome nanocarriers (NLS-NCs) and studied the NCT mechanism underlying their selective nuclear uptake. Detailed chemical, biophysical, and cellular analyses show that karyopherin receptors are required to authenticate, bind, and escort NLS-NCs through NPCs while Ran guanosine triphosphate (RanGTP) promotes their release from NPCs into the nuclear interior. Ultrastructural analysis by regressive staining transmission electron microscopy further resolves the NLS-NCs on transit in NPCs and inside the nucleus. By elucidating their ability to utilize NCT, these findings demonstrate the efficacy of polymersomes to deliver encapsulated payloads directly into cell nuclei.

Development of Polymersomes Co-Delivering Doxorubicin and Melittin to Overcome Multidrug Resistance.

Multidrug resistance (MDR) is one of the major barriers in chemotherapy. It is often related to the overexpression of efflux receptors such as P-glycoprotein (P-gp). Overexpressed efflux receptors inhibit chemotherapeutic efficacy by pumping out intracellularly delivered anticancer drugs. In P-gp-mediated MDR-related pathways, PI3K/Akt and NF-kB pathways are commonly activated signaling pathways, but these pathways are downregulated by melittin, a main component of bee venom. In this study, a polymersome based on a poly (lactic acid) (PLA)-hyaluronic acid (HA) (20k-10k) di-block copolymer and encapsulating melittin and doxorubicin was developed to overcome anticancer resistance and enhance chemotherapeutic efficacy. Through the simultaneous delivery of doxorubicin and melittin, PI3K/Akt and NF-κB pathways could be effectively inhibited, thereby downregulating P-gp and successfully enhancing chemotherapeutic efficacy. In conclusion, a polymersome carrying an anticancer drug and melittin could overcome MDR by regulating P-gp overexpression pathways.

Dually Responsive Poly(N-vinylcaprolactam)-b-poly(dimethylsiloxane)-b-poly(N-vinylcaprolactam) Polymersomes for Controlled Delivery.

Limited tissue selectivity and targeting of anticancer therapeutics in systemic administration can produce harmful side effects in the body. Various polymer nano-vehicles have been developed to encapsulate therapeutics and prevent premature drug release. Dually responsive polymeric vesicles (polymersomes) assembled from temperature-/pH-sensitive block copolymers are particularly interesting for the delivery of encapsulated therapeutics to targeted tumors and inflamed tissues. We have previously demonstrated that temperature-responsive poly(N-vinylcaprolactam) (PVCL)-b-poly(dimethylsiloxane) (PDMS)-b-PVCL polymersomes exhibit high loading efficiency of anticancer therapeutics in physiological conditions. However, the in-vivo toxicity of these polymersomes as biocompatible materials has not yet been explored. Nevertheless, developing an advanced therapeutic nanocarrier must provide the knowledge of possible risks from the material's toxicity to support its future clinical research in humans. Herein, we studied pH-induced degradation of PVCL10-b-PDMS65-b-PVCL10 vesicles in-situ and their dually (pH- and temperature-) responsive release of the anticancer drug, doxorubicin, using NMR, DLS, TEM, and absorbance spectroscopy. The toxic potential of the polymersomes was evaluated in-vivo by intravenous injection (40 mg kg-1 single dose) of PVCL10-PDMS65-PVCL10 vesicles to mice. The sub-acute toxicity study (14 days) included gravimetric, histological, and hematological analyses and provided evidence for good biocompatibility and non-toxicity of the biomaterial. These results show the potential of these vesicles to be used in clinical research.

Paper-based lateral flow assay using rhodamine B-loaded polymersomes for the colorimetric determination of synthetic cannabinoids in saliva.

Synthetic cannabinoids are one of the many substances of abuse widely spreading in modern society. Medical practitioners and law enforcement alike highly seek portable, efficient, and reliable tools for on-site detection and diagnostics. Here, we propose a colorimetric lateral flow assay (LFA) combined with dye-loaded polymersome to detect the synthetic cannabinoid JWH-073 efficiently. Rhodamine B-loaded polymersome was conjugated to antibodies and fully characterized. Two LFA were proposed (sandwich and competitive), showing a high level of sensitivity with a limit of detection (LOD) reaching 0.53 and 0.31 ng/mL, respectively. The competitive assay was further analyzed by fluorescence, where the LOD reached 0.16 ng/mL. The application of the LFA over spiked synthetic saliva or real human saliva demonstrated an overall response of 94% for the sandwich assay and 97% for the competitive LFA. The selectivity of the system was assessed in the presence of various interferents. The analytical performance of the LFA system showed a coefficient of variation below 6%. The current LFA system appears as a plausible system for non-invasive detection of substance abuse and shows promise for synthetic cannabinoid on-site sensing.

Increased Protein Encapsulation in Polymersomes with Hydrophobic Membrane Anchoring Peptides in a Scalable Process.

Hollow vesicles made from a single or double layer of block-copolymer molecules, called polymersomes, represent an important technological platform for new developments in nano-medicine and nano-biotechnology. A central aspect in creating functional polymersomes is their combination with proteins, especially through encapsulation in the inner cavity of the vesicles. When producing polymersomes by techniques such as film rehydration, significant proportions of the proteins used are trapped in the vesicle lumen, resulting in high encapsulation efficiencies. However, because of the difficulty of scaling up, such methods are limited to laboratory experiments and are not suitable for industrial scale production. Recently, we developed a scalable polymersome production process in stirred-tank reactors, but the statistical encapsulation of proteins resulted in fairly low encapsulation efficiencies of around 0.5%. To increase encapsulation in this process, proteins were genetically fused with hydrophobic membrane anchoring peptides. This resulted in encapsulation efficiencies of up to 25.68%. Since proteins are deposited on the outside and inside of the polymer membrane in this process, two methods for the targeted removal of protein domains by proteolysis with tobacco etch virus protease and intein splicing were evaluated. This study demonstrates the proof-of-principle for production of protein-functionalized polymersomes in a scalable process.

Membrane Engineering: Phase Separation in Polymeric Giant Vesicles.

Cell membranes exhibit elaborate lipidic patterning to carry out a myriad of functions such as signaling and trafficking. Domain formation in giant unilamellar vesicles (GUVs) is thus of interest for understanding fundamental biological processes and to provide new prospects for biocompatible soft materials. Lipid rearrangements in lipidic GUVs and lipid/polymer GUVs are extensively studied whereas polymer/polymer hybrid GUVs remain evasive. Here, the focus is on the thermodynamically driven phase separation of amphiphilic polymers in GUVs. It is demonstrated that polymer phase separation is entropically dictated by hydrophobic block incompatibilities and that films topology can help to determine the outcome of polymeric phase separation in GUVs. Lastly, Janus-GUVs are obtained and GUVs exhibit a single large domain by using a compatibilizing hydrophobic block copolymer.

Segregated Nanocompartments Containing Therapeutic Enzymes and Imaging Compounds within DNA-Zipped Polymersome Clusters for Advanced Nanotheranostic Platform.

Nanotheranostics is an emerging field that brings together nanoscale-engineered materials with biological systems providing a combination of therapeutic and diagnostic strategies. However, current theranostic nanoplatforms have serious limitations, mainly due to a mismatch between the physical properties of the selected nanomaterials and their functionalization ease, loading ability, or overall compatibility with bioactive molecules. Herein, a nanotheranostic system is proposed based on nanocompartment clusters composed of two different polymersomes linked together by DNA. Careful design and procedure optimization result in clusters segregating the therapeutic enzyme human Dopa decarboxylase (DDC) and fluorescent probes for the detection unit in distinct but colocalized nanocompartments. The diagnostic compartment provides a twofold function: trackability via dye loading as the imaging component and the ability to attach the cluster construct to the surface of cells. The therapeutic compartment, loaded with active DDC, triggers the cellular expression of a secreted reporter enzyme via production of dopamine and activation of dopaminergic receptors implicated in atherosclerosis. This two-compartment nanotheranostic platform is expected to provide the basis of a new treatment strategy for atherosclerosis, to expand versatility and diversify the types of utilizable active molecules, and thus by extension expand the breadth of attainable applications.

pH-bioresponsive poly(ε-caprolactone)-based polymersome for effective drug delivery in cancer and protein glycoxidation prevention.

Artificial nanostructures using polymers to produce polymeric vesicles are inspired by the many intricate structures found in living organisms. Polymersomes are a class of self-assembled vesicles known for their great stability and application in drug delivery. They can be tuned according to their intended use by changing their components and introducing activable block copolymers that transform these polymersomes into smart nanocarriers. In this study, we propose the synthesis of a poly (ethylene oxide)-poly (ε-caprolactone)-based polymersome (PEO-PCL) loaded with GSH as a pH-responsive drug delivery molecule for cancer and protein alteration inhibition. Initially, the nanocarrier was synthesized and characterized by DLS, TEM/SEM microscopy as well as gel permeation chromatography (GPC) and 1H NMR. Their CMC formation, encapsulation efficiency, and pH responsiveness were analyzed. In addition, empty and GSH-loaded PEO-PCL polymersomes were tested for their toxicity and therapeutic effect on normal and cancer cells via an MTT test. Subsequently, protein alteration models (aggregation, glycation, and oxidation) were performed in vitro where the polymersomes were tested. Results showed that other than being non-toxic and able to highly encapsulate and release the GSH in response to acidic conditions, the nanocomposites do not hinder its content's ameliorative effects on cancer cells and protein alterations. This infers that polymeric nanocarriers can be a base for future smart biomedicine applications and theranostics.

Stationary photocurrent generation from bacteriorhodopsin-loaded lipo-polymersomes in polyelectrolyte multilayer assembly on polyethersulfone membrane.

Vesicles constructed of either synthetic polymers alone (polymersomes) or a combination of polymers and lipids (lipo-polymersomes) demonstrate excellent long-term stability and ability to integrate membrane proteins. Applications using lipo-polymersomes with integrated membrane proteins require suitable supports to maintain protein functionality. Using lipo-polymersomes loaded with the light-driven proton pump bacteriorhodopsin (BR), we demonstrate here how the photocurrent is influenced by a chosen support. In our study, we deposited BR-loaded lipo-polymersomes in a cross-linked polyelectrolyte multilayer assembly either directly physisorbed on gold electrode microchips or cross-linked on an intermediary polyethersulfone (PES) membrane covalently grafted using a hydrogel cushion. In both cases, electrochemical impedance spectroscopic characterization demonstrated successful polyelectrolyte assembly with BR-loaded lipo-polymersomes. Light-induced proton pumping by BR-loaded lipo-polymersomes in the different support constructs was characterized by amperometric recording of the generated photocurrent. Application of the hydrogel/PES membrane support together with the polyelectrolyte assembly decreased the transient current response upon light activation of BR, while enhancing the generated stationary current to over 700 nA/cm2. On the other hand, the current response from BR-loaded lipo-polymersomes in a polyelectrolyte assembly without the hydrogel/PES membrane support was primarily a transient peak combined with a low-nanoampere-level stationary photocurrent. Hence, the obtained results demonstrated that by using a hydrogel/PES support it was feasible to monitor continuously light-induced proton flux in biomimetic applications of lipo-polymersomes. Graphical abstract.

Synthesis of chimeric polymersomes based on PLA-b-PHPMA and PCL-b-PHPMA for nucleoline guided delivery of SN38.

We reported SN38-loaded polymersomes formulated with amphiphilic block copolymers based on HPMA and either ε-caprolactone or lactic acid through employing ring-opening polymerization, carbodiimide chemistry and a reversible addition-fragmentation chain transfer polymerization technique. In this regard, we successfully synthesized five chimeric polymersomes based on different percentage of the synthesized copolymers. The prepared chimeric polymersomes based on PCL-b-PHPMA:PLA-b-PHPMA at ratio of 1:3 exhibited superior loading capacity in comparison with other chimeric polymersomes. In order to increase therapeutic index of the prepared systems, AS1411 aptamer was implemented as targeting ligand. In vivo study revealed that the intravenous single dose injection of targeted chimeric polymersomes to C26 tumor bearing mice had remarkable efficacy in inhibiting tumor growth. It could be concluded that the chimeric polymersomes fabricated from PCL-b-PHPMA and PLA-b-PHPMA at a ratio of 1:3 have great potential for SN38 encapsulation while providing controlled sustained release properties with targeting capability via AS1411 aptamer conjugation.

Surface engineered polymersomes for enhanced modulation of dendritic cells during cardiovascular immunotherapy.

The principle cause of cardiovascular disease (CVD) is atherosclerosis, a chronic inflammatory condition characterized by immunologically complex fatty lesions within the intima of arterial vessel walls. Dendritic cells (DCs) are key regulators of atherosclerotic inflammation, with mature DCs generating pro-inflammatory signals within vascular lesions and tolerogenic DCs eliciting atheroprotective cytokine profiles and regulatory T cell (Treg) activation. Here, we engineered the surface chemistry and morphology of synthetic nanocarriers composed of poly(ethylene glycol)-b-poly(propylene sulfide) copolymers to selectively target and modulate DCs by transporting the anti-inflammatory agent 1, 25-Dihydroxyvitamin D3 (aVD) and ApoB-100 derived antigenic peptide P210. Polymersomes decorated with an optimized surface display and density for a lipid construct of the P-D2 peptide, which binds CD11c on the DC surface, significantly enhanced the cytosolic delivery and resulting immunomodulatory capacity of aVD in vitro. Intravenous administration of the optimized polymersomes achieved selective targeting of DCs in atheroma and spleen compared to all other cell populations, including both immune and CD45- cells, and locally increased the presence of tolerogenic DCs and cytokines. aVD-loaded polymersomes significantly inhibited atherosclerotic lesion development in high fat diet-fed ApoE-/- mice following 8 weeks of administration. Incorporation of the P210 peptide generated the largest reductions in vascular lesion area (~33%, p<0.001), macrophage content (~55%, p<0.001), and vascular stiffness (4.8-fold). These results correlated with an ~6.5-fold increase in levels of Foxp3+ regulatory T cells within atherosclerotic lesions. Our results validate the key role of DC immunomodulation during aVD-dependent inhibition of atherosclerosis and demonstrate the therapeutic enhancement and dosage lowering capability of cell-targeted nanotherapy in the treatment of CVD.

Antibacterial effects of curcumin encapsulated in nanoparticles on clinical isolates of Pseudomonas aeruginosa through downregulation of efflux pumps.

Curcumin as a flavonoid from the rhizome of Curcuma longa has antibacterial, antiviral and antifungal activity. Multidrug resistance in pathogenic bacteria is continuously increasing in hospitals. The aim of this study was to investigate the effect of curcumin encapsulated in micellar/polymersome nanoparticles as an efflux pump inhibitor (EPI) on the expression of mexX and oprM genes in curcumin-treated and -untreated isolates of Pseudomonas aeruginosa. Clinical isolates of Pseudomonas aeruginosa were treated with ciprofloxacin (sub-MICs) alone and/or in combination with curcumin-encapsulated in micellar/polymersome nanoparticles. The expression of mexX and oprM genes was quantitatively evaluated by qRT-PCR in curcumin-treated and -untreated bacteria after 24 h. Curcumin-encapsulated in nanoparticles (400 µg/mL) induced cell death up to 50% in ciprofloxacin-treated (1/2MIC) resistant isolates during 24 h, while the bacteria treated with ciprofloxacin (without curcumin) were not inhibited. Also, curcumin in different concentrations increased effect of ciprofloxacin (sub-MICs). Downregulation of mexX and oprM genes was observed in cells treated with curcumin and ciprofloxacin compared to cells treated with ciprofloxacin alone. It seems that curcumin can be used as complementary drug in ciprofloxacin-resistant isolates through downregulating genes involved in efflux pumps and trapping ciprofloxacin on bacterial cells and increasing the effects of drug.

Stomatocyte in Stomatocyte: A New Shape of Polymersome Induced via Chemical-Addition Methodology.

Accurate control of the shape transformation of polymersome is an important and interesting challenge that spans across disciplines such as nanomedicine and nanomachine. Here, we report a fast and facile methodology of shape manipulation of polymersome via out-of-equilibrium polymer self-assembly and shape change by chemical addition of additives. Due to its increased permeability, hydrophilicity, and fusogenic properties, poly(ethylene oxide) was selected as the additive for bringing the system out of equilibrium via fast addition into the polymersome organic solution. A new shape, stomatocyte-in-stomatocyte (sto-in-sto), is obtained for the first time. Moreover, fast shape transformation within less than 1 min to other relevant shapes such as stomatocyte and large compound vesicles was also obtained and accurately controlled in a uniform dispersion. This methodology is demonstrated as a general strategy with which to push the assembly further out of equilibrium to generate unusual nanostructures in a controllable and fast manner.

Enzymatically Powered Surface-Associated Self-Motile Protocells.

Cell motility is central to processes such as wound healing, immune cell surveillance, and embryonic development. Motility requires the conversion of chemical to mechanical energy. An active area of research is to create motile particles, such as microswimmers, using catalytic and enzymatic reactions. Here, autonomous motion is demonstrated in adhesive polymer-based protocells by incorporating and harnessing the energy production of an enzymatic reaction. Biotinylated polymer vesicles that encapsulate catalase, an enzyme which converts hydrogen peroxide to water and oxygen, are prepared and these vesicles are adhered weakly to avidin-coated surfaces. Upon addition of hydrogen peroxide, which diffuses across the membrane, catalase activity generates a differential impulsive force that enables the breakage and reformation of biotin-avidin bonds, leading to diffusive vesicle motion resembling random motility. The random motility requires catalase, increases with the concentration of hydrogen peroxide, and needs biotin-avidin adhesion. Thus, a protocellular mimetic of a motile cell.

Nanosensors based on polymer vesicles and planar membranes: a short review.

This review aims to summarize the advance in the field of nanosensors based on two particular materials: polymer vesicles (polymersomes) and polymer planar membranes. These two types of polymer-based structural arrangements have been shown to be efficient in the production of sensors as their features allow to adapt to different environment but also to increase the sensitivity and the selectivity of the sensing device. Polymersomes and planar polymer membranes offer a platform of choice for a wide range of chemical functionalization and characteristic structural organization which allows a convenient usage in numerous sensing applications. These materials appear as great candidates for such nanosensors considering the broad variety of polymers. They also enable the confection of robust nanosized architectures providing interesting properties for numerous applications in many domains ranging from pollution to drug monitoring. This report gives an overview of these different sensing strategies whether the nanosensors aim to detect chemicals, biological or physical signals.

Clinical ultrasound stimulating angiogenesis following drug-release from polymersomes on the ischemic zone for peripheral arterial occlusive disease.

Peripheral Arterial Occlusive Disease (PAOD) is an aging disease that affects the quality of life of many people by its intermittent claudication and critical limb ischemia presentations. Traditional treatment and management of PAOD are asking patients to make a life change and medication with antiplatelet, statins and cilostazol, which decrease the possibility of clot formation. Our strategy has employed a magnetic Fe3O4-PLGA polymersome to carry the cilostazol into the ischemic area by magnetic attraction following remote-control drug release through low-energy ultrasound exposure. In the animal studies, the cilostazol-loaded Fe3O4-PLGA polymersomes were injected and accumulated at ischemic leg through magnetic attraction. Then, using a clinical-use ultrasound machine the leg was irradiated to forward cilostazol release from the accumulated polymersomes. Dramatically, we found an observable result of bloody flux recovery in the leg after 7 days compared to the non-treated leg that showed no evidence of the blood recovery.

Toll-like receptor 8 agonist nanoparticles mimic immunomodulating effects of the live BCG vaccine and enhance neonatal innate and adaptive immune responses.

BACKGROUND: Newborns display distinct immune responses, leaving them vulnerable to infections and impairing immunization. Targeting newborn dendritic cells (DCs), which integrate vaccine signals into adaptive immune responses, might enable development of age-specific vaccine formulations to overcome suboptimal immunization. OBJECTIVE: Small-molecule imidazoquinoline Toll-like receptor (TLR) 8 agonists robustly activate newborn DCs but can result in reactogenicity when delivered in soluble form. We used rational engineering and age- and species-specific modeling to construct and characterize polymer nanocarriers encapsulating a TLR8 agonist, allowing direct intracellular release after selective uptake by DCs. METHODS: Chemically similar but morphologically distinct nanocarriers comprised of amphiphilic block copolymers were engineered for targeted uptake by murine DCs in vivo, and a range of TLR8 agonist-encapsulating polymersome formulations were then synthesized. Novel 96-well in vitro assays using neonatal human monocyte-derived DCs and humanized TLR8 mouse bone marrow-derived DCs enabled benchmarking of the TLR8 agonist-encapsulating polymersome formulations against conventional adjuvants and licensed vaccines, including live attenuated BCG vaccine. Immunogenicity of the TLR8 agonist adjuvanted antigen 85B (Ag85B)/peptide 25-loaded BCG-mimicking nanoparticle formulation was evaluated in vivo by using humanized TLR8 neonatal mice. RESULTS: Although alum-adjuvanted vaccines induced modest costimulatory molecule expression, limited TH-polarizing cytokine production, and significant cell death, BCG induced a robust adult-like maturation profile of neonatal DCs. Remarkably, TLR8 agonist polymersomes induced not only newborn DC maturation profiles similar to those induced by BCG but also stronger IL-12p70 production. On subcutaneous injection to neonatal mice, the TLR8 agonist-adjuvanted Ag85B peptide 25 formulation was comparable with BCG in inducing Ag85B-specific CD4+ T-cell numbers. CONCLUSION: TLR8 agonist-encapsulating polymersomes hold substantial potential for early-life immunization against intracellular pathogens. Overall, our study represents a novel approach for rational design of early-life vaccines.