Programming of in Situ Tumor Vaccines via Supramolecular Nanodrug/Hydrogel Composite and Deformable Nanoadjuvant for Cancer Immunotherapy.

The development of in situ tumor vaccines offers promising prospects for cancer treatment. Nonetheless, the generation of plenary autologous antigens in vivo and their codelivery to DC cells along with adjuvants remains a significant challenge. Herein, we developed an in situ tumor vaccine using a supramolecular nanoparticle/hydrogel composite (ANPMTO/ALCD) and a deformable nanoadjuvant (PPER848). The ANPMTO/ALCD composite consisted of ß-cyclodextrin-decorated alginate (Alg-g-CD) and MTO-encapsulated adamantane-decorated nanoparticles (ANPMTO) through supramolecular interaction, facilitating the long-term and sustained production of plenary autologous antigens, particularly under a 660 nm laser. Simultaneously, the produced autologous antigens were effectively captured by nanoadjuvant PPER848 and subsequently transported to lymph nodes and DC cells, benefiting from its optimized size and deformability. This in situ tumor vaccine can trigger a robust antitumor immune response and demonstrate significant therapeutic efficacy in inhibiting tumor growth, suppressing tumor metastasis, and preventing postoperative recurrence, offering a straightforward approach to programming in situ tumor vaccines.

Genome-centric metagenomics and methanogenic pathway analysis for acclimated anaerobic digestion of chicken manure with high ammonia stressed under thermophilic condition.

Thermophilic anaerobic digestion (AD) of animal manure offers various environmental benefits but the process requires a microbial community acclimatized to high ammonia. In current study, a lab-scale continuous stirred tank reactor (CSTR) fed with chicken manure was operated under thermophilic condition for 450 days in total. Results showed that the volumetric methane production decreased from 445 to 328 and sharply declined to 153 mL L-1·d-1 with feeding total solid (TS) step increased from 5% to 7.5% and 10%, respectively. While, after a long-term stop feeding for 80 days, highly disturbed reactor was able to recover methane generation to 739 mL L-1·d-1 at feeding TS of 10%. Isotope analysis indicted acetate converted to methane through the syntrophic acetate oxidation and hydrogenotrophic methanogenesis (SAO-HM) pathway increased from 33% to 63% as the concentration of ammonium increased from 2493 to 6258 mg L-1. Significant different in the genome expression of the SAO bacterial from 0.09% to 1.23%, combining with main hydrogenotrophic partners (Methanoculleus spp. and Methanothermobacter spp.) contented of 2.1% and 99.9% during inhibitory and recovery stages, respectively. The highly expressed KEGG pathway in level 3 (enzyme genes) for the Recovery sludge combining with the extraordinary high abundance of genera Halocella sp. suggested that Halocella sp. might be a highly efficient hydrolytic and acidogenic microorganism and enhance the process of SAO during carbon metabolic flow to methane. This report will be a basis for further study of AD studies on high nitrogen content of poultry manure.

Optimized Cationic Lipid-assisted Nanoparticle for Delivering CpG Oligodeoxynucleotides to Treat Hepatitis B Virus Infection.

PURPOSE: Hepatitis B virus (HBV) infection is such a global health problem that hundreds of millions of people are HBV carriers. Current anti-viral agents can inhibit HBV replication, but can hardly eradicate HBV. Cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODNs) are an adjuvant that can activate plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) to induce therapeutic immunity for HBV eradication. However, efficient delivery of CpG ODNs into pDCs and cDCs remains a challenge. In this study, we constructed a series of cationic lipid-assisted nanoparticles (CLANs) using different cationic lipids to screen an optimal nanoparticle for delivering CpG ODNs into pDCs and cDCs. METHODS: We constructed different CLANCpG using six cationic lipids and analyzed the cellular uptake of different CLANCpG by pDCs and cDCs in vitro and in vivo, and further analyzed the efficiency of different CLANCpG for activating pDCs and cDCs in both wild type mice and HBV-carrier mice. RESULTS: We found that CLAN fabricated with 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP) showed the highest efficiency for delivering CpG ODNs into pDCs and cDCs, resulting in strong therapeutic immunity in HBV-carrier mice. By using CLANCpG as an immune adjuvant in combination with the injection of recombinant hepatitis B surface antigen (rHBsAg), HBV was successfully eradicated and the chronic liver inflammation in HBV-carrier mice was reduced. CONCLUSION: We screened an optimized CLAN fabricated with DOTAP for efficient delivery of CpG ODNs to pDCs and cDCs, which can act as a therapeutic vaccine adjuvant for treating HBV infection.

Generalized Oscillator Strengths for the Valence Shell Excitations in Carbon Tetrachloride Studied by Fast Electron Impact.

A joint experimental and theoretical investigation of the valence shell excitations of carbon tetrachloride has been performed by fast electron scattering and time dependent density functional theory calculations. At a collision energy of 1.5 keV and an energy resolution of about 70 meV, the dipole-forbidden transition of a1σ* ← 2t1 has been clearly observed at large momentum transfers, and its excitation energy of 6.15 eV and line width of 0.72 eV have been determined. Two new features are also recognized at 9.97 and 10.26 eV. The generalized oscillator strengths of the excited states at 5-11.3 eV have been determined from the measured spectra. The calculated generalized oscillator strength of the a1σ* ← 2t1 transition with the vibronic effect shows better agreement with the experiment, and the vibronic effect also accounts for its nonzero intensity at zero squared momentum transfer. The optical oscillator strengths of the valence shell excitations have also been obtained by extrapolating the generalized oscillator strengths to the limit of zero squared momentum transfer. The integral cross sections have been systematically determined from the threshold to 5000 eV by means of the BE-scaling method. The present oscillator strengths and cross sections provide the fundamental data of carbon tetrachloride and have important applications in photochemical modeling for atmospheric physics.

The Study of the Low-Lying Valence-Shell Excitations of Hydrogen Sulfide by Fast Electron Impact.

The valence-shell excitations of hydrogen sulfide have been studied by fast electron impact at a collision energy of 1.5 keV and an energy resolution of about 70 meV. By analyzing the variations of intensity and shape of the feature in the range of 5.0-7.5 eV at different scattering angles, the excitation energy of 5.85 ± 0.01 eV and the line width of 0.80 ± 0.01 eV of the 3b21A2 state have been determined. The generalized oscillator strengths of the valence-shell excitations in the energy range of 5.0-9.2 eV of hydrogen sulfide have been determined from the measured spectra. The corresponding optical oscillator strengths have been obtained by extrapolating the generalized oscillator strengths to the limit of zero squared momentum transfer. The integral cross sections have also been systematically determined from the threshold to 5000 eV by means of the BE-scaling method. The presently obtained oscillator strengths and integral cross sections have significant applications in the studies of planetary atmospheres and interstellar gases.

Oscillator strengths and integral cross sections of the valence-shell excitations of HCl studied by fast electron scattering.

The oscillator strengths and integral cross sections of the valence-shell excitations of HCl have significant applications in the studies of planetary atmospheres and interstellar gases. In the present work, the generalized oscillator strengths of the valence-shell excitations of HCl have been measured at an incident electron energy of 1500 eV and an energy resolution of 70 meV, and their momentum transfer dependence behaviors have been elucidated. It is observed that the generalized oscillator strength ratios of the b3Π1(ν' = 0) state to the C1Π(ν' = 0) state are a constant and independent of the squared momentum transfer, and this typical behavior in the momentum space is explained by the intraconfiguration mixing of the b3Π1 and C1Π states due to the spin-orbital interaction. The optical oscillator strengths of the valence-shell excitations have been obtained by extrapolating the generalized oscillator strengths to the limit of zero squared momentum transfer. The present optical oscillator strengths give an independent cross-check to the previous experimental and theoretical results, and it is found that most of the photoabsorption measurements are limited by the line saturation effect. The integral cross sections of the valence-shell excitations of HCl have been obtained systematically from the threshold to 5000 eV with the aid of the BE-scaling method.

Oscillator strengths and integral cross sections of the ÃA2″1← X̃1A1 excitation of ammonia studied by fast electron impact.

The vibrationally resolved generalized oscillator strengths of the first and strongest singlet excitation ÃA2″1← X̃1A1 of ammonia have been determined at an impact electron energy of 1500 eV with an energy resolution of 80 meV. The comprehensive comparison of the present results with the previous experimental and theoretical ones shows that the high-energy limit, where the first Born approximation holds, has been reached at an impact electron energy of 1500 eV in K2 < 1 a.u., while it is still not satisfied in the K2 > 1 a.u. even at 1500 eV. It is also observed that the minimum position of the generalized oscillator strength of the vibronic state shifts toward the larger squared momentum transfer with the increasing vibrational quantum number. By extrapolating the generalized oscillator strength to the zero momentum transfer, the optical oscillator strength of the ÃA2″1 state has been obtained, which gives an independent cross check to the previous results. The integral cross sections of the ÃA2″1 state have been obtained systematically from the threshold to 5000 eV with the aid of the BE-scaling method.

Stimuli-responsive clustered nanoparticles for improved tumor penetration and therapeutic efficacy.

A principal goal of cancer nanomedicine is to deliver therapeutics effectively to cancer cells within solid tumors. However, there are a series of biological barriers that impede nanomedicine from reaching target cells. Here, we report a stimuli-responsive clustered nanoparticle to systematically overcome these multiple barriers by sequentially responding to the endogenous attributes of the tumor microenvironment. The smart polymeric clustered nanoparticle (iCluster) has an initial size of ∼100 nm, which is favorable for long blood circulation and high propensity of extravasation through tumor vascular fenestrations. Once iCluster accumulates at tumor sites, the intrinsic tumor extracellular acidity would trigger the discharge of platinum prodrug-conjugated poly(amidoamine) dendrimers (diameter ∼5 nm). Such a structural alteration greatly facilitates tumor penetration and cell internalization of the therapeutics. The internalized dendrimer prodrugs are further reduced intracellularly to release cisplatin to kill cancer cells. The superior in vivo antitumor activities of iCluster are validated in varying intractable tumor models including poorly permeable pancreatic cancer, drug-resistant cancer, and metastatic cancer, demonstrating its versatility and broad applicability.

Acid-Activatable Michael-Type Fluorescent Probes for Thiols and for Labeling Lysosomes in Live Cells.

A Michael addition is usually taken as a base-catalyzed reaction. Most fluorescent probes have been designed to detect thiols in slightly alkaline solutions (pH 7-9). The sensing reactions of almost all Michael-type fluorescent probes for thiols are faster in a high pH solution than in a low pH solution. In this work, we synthesized a series of 7-substituted 2-(quinolin-2-ylmethylene)malonic acids (QMAs, substituents: NEt2, OH, H, Cl, or NO2) and their ethyl esters (QMEs) as Michael-type fluorescent probes for thiols. The sensing reactions of QMAs and QMEs occur in distinct pH ranges, pH < 7 for QMAs and pH > 7 for QMEs. On the basis of experimental and theoretic studies, we have clarified the distinct pH effects on the sensing reactivity between QMAs and QMEs and demonstrated that two QMAs (NEt2, OH) are highly sensitive and selective fluorescent probes for thiols in acidic solutions (pH < 7) and promising dyes that can label lysosomes in live cells.

Synthetic lethal therapy for KRAS mutant non-small-cell lung carcinoma with nanoparticle-mediated CDK4 siRNA delivery.

The KRAS mutation is present in ~20% of lung cancers and has not yet been effectively targeted for therapy. This mutation is associated with a poor prognosis in non-small-cell lung carcinomas (NSCLCs) and confers resistance to standard anticancer treatment drugs, including epidermal growth factor receptor tyrosine kinase inhibitors. In this study, we exploited a new therapeutic strategy based on the synthetic lethal interaction between cyclin-dependent kinase 4 (CDK4) downregulation and the KRAS mutation to deliver micellar nanoparticles (MNPs) containing small interfering RNA targeting CDK4 (MNPsiCDK4) for treatment in NSCLCs harboring the oncogenic KRAS mutation. Following MNPsiCDK4 administration, CDK4 expression was decreased, accompanied by inhibited cell proliferation, specifically in KRAS mutant NSCLCs. However, this intervention was harmless to normal KRAS wild-type cells, confirming the proposed mechanism of synthetic lethality. Moreover, systemic delivery of MNPsiCDK4 significantly inhibited tumor growth in an A549 NSCLC xenograft murine model, with depressed expression of CDK4 and mutational KRAS status, suggesting the therapeutic promise of MNPsiCDK4 delivery in KRAS mutant NSCLCs via a synthetic lethal interaction between KRAS and CDK4.

Cationic lipid-assisted polymeric nanoparticle mediated GATA2 siRNA delivery for synthetic lethal therapy of KRAS mutant non-small-cell lung carcinoma.

Synthetic lethal interaction provides a conceptual framework for the development of wiser cancer therapeutics. In this study, we exploited a therapeutic strategy based on the interaction between GATA binding protein 2 (GATA2) downregulation and the KRAS mutation status by delivering small interfering RNA targeting GATA2 (siGATA2) with cationic lipid-assisted polymeric nanoparticles for treatment of non-small-cell lung carcinoma (NSCLC) harboring oncogenic KRAS mutations. Nanoparticles carrying siGATA2 (NPsiGATA2) were effectively taken up by NSCLC cells and resulted in targeted gene suppression. NPsiGATA2 selectively inhibited cell proliferation and induced cell apoptosis in KRAS mutant NSCLC cells. However, this intervention was harmless to normal KRAS wild-type NSCLC cells and HL7702 hepatocytes, confirming the advantage of synthetic lethality-based therapy. Moreover, systemic delivery of NPsiGATA2 significantly inhibited tumor growth in the KRAS mutant A549 NSCLC xenograft murine model, suggesting the therapeutic promise of NPsiGATA2 delivery in KRAS mutant NSCLC therapy.

Ratiometric two-photon fluorescent probes for mitochondrial hydrogen sulfide in living cells.

Hydrogen sulfide (H2S) is an important signaling molecule with diverse biological roles. Various fluorescent probes for H2S with biological application have been developed. However, two-photon ratiometric imaging of mitochondrial H2S is scarce. In this paper, we report two ratiometric two-photon probes, AcHS-1 and AcHS-2, which employ 4-amino-1,8-naphthalimide as the fluorophore and 4-azidobenzyl carbamate as the H2S response site. These probes exhibit high selectivity toward H2S over biothiols and other reactive species, low detection limits of 50-85 nM, low cytotoxicity, and high stability under physiological conditions. Furthermore, through cell imaging with one-photon and two-photon microscopy, MCF-7 cells incubated with two probes show a marked change in emission color from blue to green in response to H2S. Cell images costraining with a mitochondrial dye reveal that AcHS-2 is a mitochondria-specific two-photon probe for H2S. These results show that AcHS-2 may find useful applications in biological research such as tracking mitochondrial H2S in living biological specimens.

Delivery of mRNA for regulating functions of immune cells.

Abnormal immune cell functions are commonly related to various diseases, including cancer, autoimmune diseases, and infectious diseases. Messenger RNA (mRNA)-based therapy can regulate the functions of immune cells or assign new functions to immune cells, thereby generating therapeutic immune responses to treat these diseases. However, mRNA is unstable in physiological environments and can hardly enter the cytoplasm of target cells; thus, effective mRNA delivery systems are critical for developing mRNA therapy. The two mRNA vaccines of Pfizer-BioNTech and Moderna have demonstrated that lipid nanoparticles (LNPs) can deliver mRNA into dendritic cells (DCs) to induce immunization against severe acute respiratory syndrome coronavirus 2, which opened the floodgates to the development of mRNA therapy. Apart from DCs, other immune cells are promising targets for mRNA therapy. This review summarized the barriers to mRNA delivery and advances in mRNA delivery for regulating the functions of different immune cells.

Tailor-made dual pH-sensitive polymer-doxorubicin nanoparticles for efficient anticancer drug delivery.

Efficient delivery of therapeutics into tumor cells to increase the intracellular drug concentration is a major challenge for cancer therapy due to drug resistance and inefficient cellular uptake. Herein, we have designed a tailor-made dual pH-sensitive polymer-drug conjugate nanoparticulate system to overcome the challenges. The nanoparticle is capable of reversing its surface charge from negative to positive at tumor extracellular pH (∼6.8) to facilitate cell internalization. Subsequently, the significantly increased acidity in subcellular compartments such as the endosome (∼5.0) further promotes doxorubicin release from the endocytosed drug carriers. This dual pH-sensitive nanoparticle has showed enhanced cytotoxicity in drug-resistant cancer stem cells, indicating its great potential for cancer therapy.

An All-in-One Nanomedicine Consisting of CRISPR-Cas9 and an Autoantigen Peptide for Restoring Specific Immune Tolerance.

Nanotechnology has shown great promise in treating diverse diseases. However, developing nanomedicines that can cure autoimmune diseases without causing systemic immunosuppression is still quite challenging. Herein, we propose an all-in-one nanomedicine comprising an autoantigen peptide and CRISPR-Cas9 to restore specific immune tolerance by engineering dendritic cells (DCs) into a tolerogenic phenotype, which can expand autoantigen-specific regulatory T (Treg) cells. In brief, we utilized cationic lipid-assisted poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles to simultaneously encapsulate an autoimmune diabetes-relevant peptide (2.5mi), a CRISPR-Cas9 plasmid (pCas9), and three guide RNAs (gRNAs) targeting costimulatory molecules (CD80, CD86, and CD40). We demonstrated that the all-in-one nanomedicine was able to effectively codeliver these components into DCs, followed by simultaneous disruption of the three costimulatory molecules and presentation of the 2.5mi peptide on the genome-edited DCs. The resulting tolerogenic DCs triggered the generation and expansion of autoantigen-specific Treg cells by presenting the 2.5mi peptide to CD4+ T cells in the absence of costimulatory signals. Using autoimmune type 1 diabetes (T1D) as a typical disease model, we demonstrated that our nanomedicine prevented autoimmunity to islet components and inhibited T1D development. Our all-in-one nanomedicine achieved codelivery of CRISPR-Cas9 and the peptide to DCs and could be easily applied to other autoimmune diseases by substitution of different autoantigen peptides.

Incorporation of a rhodamine B conjugated polymer for nanoparticle trafficking both in vitro and in vivo.

Polymeric nanoparticles as drug delivery systems have the potential to improve the therapeutic efficacy and reduce the toxicity of chemotherapeutic drugs by enhancing the drug selectivity in vivo. The efficacy is directly dependent on the polymeric nanoparticles' in vivo fate. Therefore, it is very important to develop a method to stably label the polymeric nanoparticles for detecting the in vivo fate. Here, we report a method to stably label self-assembled nanoparticles by the incorporation of rhodamine B-conjugated poly(ε-caprolactone) (PCL-RhoB). Only 1% of PCL-RhoB was released from the RhoB-labeled polymeric nanoparticles (RhoB-PNPs) in phosphate buffer within 12 hours, which suggested that the signal of PCL-RhoB can be used to represent the behaviors of polymeric nanoparticles both in vitro and in vivo. PCL-RhoB could be effectively extracted and quantitatively detected by ultra-high-performance liquid chromatography (UPLC) in various media, such as PBS, a cell culture medium containing 10% FBS (pH = 7.4 and pH = 6.8), mouse serum, simulated intestinal fluid and cell or tissue lysis. The intracellular contents of PCL-RhoB in MDA-MB-231 cells detected by UPLC were linearly correlated to the concentration of the RhoB-PNPs. In addition, the contents of PCL-RhoB in plasma and the spleen were proportional to the injected dose of RhoB-PNPs in vivo. As an application example, the pharmacokinetics and biodistribution of the nanoparticles over time in vivo were analyzed following intravenous injection to confirm the feasibility of this method.

Enhanced Primary Tumor Penetration Facilitates Nanoparticle Draining into Lymph Nodes after Systemic Injection for Tumor Metastasis Inhibition.

Lymph nodes (LNs) are normally the primary site of tumor metastasis, and effective delivery of chemotherapeutics into LNs through systemic administration is critical for metastatic cancer treatment. Here, we uncovered that improved perfusion in a primary tumor facilitates nanoparticle translocation to LNs for inhibiting tumor metastasis. On the basis of our finding that an iCluster platform, which undergoes size reduction from ∼100 nm to ∼5 nm at the tumor site, markedly improved particle perfusion in the interstitium of the primary tumor, we further revealed in the current study that such tumor-specific size transition promoted particle intravasation into tumor lymphatics and migration into LNs. Quantitative analysis indicated that the drug deposition in LNs after iCluster treatment was significantly higher in the presence of a primary tumor in comparison with that after primary tumor resection. Early intervention of metastatic 4T1 tumors with iCluster chemotherapy and subsequent surgical resection of the primary tumor resulted in significantly extending animal survival, with 4 out of the 10 mice remaining completely tumor-free for 110 days. Additionally, in the more clinical relevant late metastatic model, iCluster inhibited the metastatic colonies to the lungs and extended animal survival time. This finding provides insights into the design of more effective nanomedicines for treating metastatic cancer.

Redox-sensitive dendrimersomes assembled from amphiphilic Janus dendrimers for siRNA delivery.

The development of delivery systems for small interfering RNA (siRNA) plays a key role in its clinical application. As the major delivery systems for siRNA, cationic polymer- or lipid-based vehicles are plagued by inherent issues. As proof of concept, a disulfide bond-containing amphiphilic Janus dendrimer (ssJD), which could be conveniently synthesized and readily scaled up with high reproducibility, was explored as a siRNA delivery system to circumvent these issues. The cationic hydrophilic head of this Janus dendrimer ensured strong and stable binding with negatively charged siRNA via electrostatic interactions, and the loaded siRNA was rapidly released from the obtained complexes under a redox environment. Therefore, after efficient internalization into tumor cells, redox-sensitive dendrimersome (RSDs)/siRNA exhibited significantly improved gene silencing efficacy.

Ultrafast charge-conversional nanocarrier for tumor-acidity-activated targeted drug elivery.

Nanocarriers with tumor-acidity-activated charge-conversional ability are of particular interest for targeted drug delivery in the field of precision nanomedicine. Nevertheless, the key challenge of this strategy is the slowness of reversing the surface charge at the tumor tissue. As a proof-of-concept, we synthesized the amphiphilic triblock polymer poly(ethylene glycol)-block-poly(2-carboxyethylacrylate)-block-poly(2-azepaneethylmethacrylate) (PEG-b-PCEA-b-PAEMA) to prepare the cisplatin-loaded nanocarrier UCC-NP/Pt. The PAEMA block at the physiological pH values was hydrophobic, which formed the core of UCC-NP/Pt. In contrast, at the tumor acidity, the tertiary amine groups of PAEMA block rapidly protonated, resulting in the ultrafast charge conversion of UCC-NP/Pt within 10 s. Such ultrafast charge-conversional effect more efficiently enhanced tumor cell internalization of nanocarriers, thus achieving targeted drug delivery, which in turn exhibited superior anticancer efficacy even in the cisplatin-resistant cells. This approach provides new avenues for tumor-acidity-activated targeted drug delivery.

The effect of surface poly(ethylene glycol) length on in vivo drug delivery behaviors of polymeric nanoparticles.

Engineering nanoparticles of reasonable surface poly(ethylene glycol) (PEG) length is important for designing efficient drug delivery systems. Eliminating the disturbance by other nanoproperties, such as size, PEG density, etc., is crucial for systemically investigating the impact of surface PEG length on the biological behavior of nanoparticles. In the present study, nanoparticles with different surface PEG length but similar other nanoproperties were prepared by using poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) copolymers of different molecular weights and incorporating different contents of PCL3500 homopolymer. The molecular weight of PEG block in PEG-PCL was between 3400 and 8000 Da, the sizes of nanoparticles were around 100 nm, the terminal PEG density was controlled at 0.4 PEG/nm2 (or the frontal PEG density was controlled at 0.16 PEG/nm2). Using these nanoproperties well-designed nanoparticles, we demonstrated PEG length-dependent changes in the biological behaviors of nanoparticles and exhibited nonmonotonic improvements as the PEG molecular weight increased from 3400 to 8000 Da. Moreover, under the experimental conditions, we found nanoparticles with a surface PEG length of 13.8 nm (MW = 5000 Da) significantly decreased the absorption with serum protein and interaction with macrophages, which led to prolonged blood circulation time, enhanced tumor accumulation and improved antitumor efficacy. The present study will help to establish a relatively precise relationship between surface PEG length and the in vivo behavior of nanoparticles.