A Thermosensitive Bi-Adjuvant Hydrogel Triggers Epitope Spreading to Promote the Anti-Tumor Efficacy of Frameshift Neoantigens.

Tumor-specific frameshift mutations encoding peptides (FSPs) are highly immunogenic neoantigens for personalized cancer immunotherapy, while their clinical efficacy is limited by immunosuppressive tumor microenvironment (TME) and self-tolerance. Here, a thermosensitive hydrogel (FSP-RZ-BPH) delivering dual adjuvants R848 (TLR7/8 agonist) + Zn2+ (cGAS-STING agonist) is designed to promote the efficacy of FSPs on murine forestomach cancer (MFC). After peritumoral injection, FSP-RZ-BPH behaves as pH-responsive sustained drug release at sites near the tumor to effectively transform the immunosuppressive TME into an inflammatory type. FSP-RZ-BPH orchestrates innate and adaptive immunity to activate dendritic cells in tumor-draining lymph nodes and increase the number of FSPs-reactive effector memory T cells (TEM) in tumor by 2.9 folds. More importantly, these TEM also exhibit memory responses to nonvaccinated neoantigens on MFC. This epitope spreading effect contributes to reduce self-tolerance to maintain long-lasting anti-tumor immunity. In MFC suppressive model, FSP-RZ-BPH achieves 84.8% tumor inhibition rate and prolongs the survival of tumor-bearing mice with 57.1% complete response rate. As a preventive tumor vaccine, FSP-RZ-BPH can also significantly delay tumor growth. Overall, the work identifies frameshift MFC neoantigens for the first time and demonstrates the thermosensitive bi-adjuvant hydrogel as an effective strategy to boost bystander anti-tumor responses of frameshift neoantigens.

A novel dual-function SERS-based identification strategy for preliminary screening and accurate diagnosis of circulating tumor cells.

Non-specific adsorption of bioprobes based on surface-enhanced Raman spectroscopy (SERS) technology inevitably endows white blood cells (WBC) in the peripheral blood with Raman signals, which greatly interfere the identification accuracy of circulating tumor cells (CTCs). In this study, an innovative strategy was proposed to effectively identify CTCs by using SERS technology assisted by a receiver operating characteristic (ROC) curve. Firstly, a magnetic Fe3O4-Au complex SERS bioprobe was developed, which could effectively capture the triple negative breast cancer (TNBC) cells and endow the tumor cells with distinct SERS signals. Then, the ROC curve obtained based on the comparison of SERS intensity of TNBC cells and WBC was used to construct a tumor cell identification model. The merit of the model was that the detection sensitivity and specificity could be intelligently switched according to different identification purposes such as accurate diagnosis or preliminary screening of tumor cells. Finally, the difunctional recognition ability of the model for accurate diagnosis and preliminary screening of tumor cells was further validated by using the healthy human blood added with TNBC cells and blood samples of real tumor patients. This novel difunctional identification strategy provides a new perspective for identification of CTCs based on the SERS technology.

Stimuli-Responsive Codelivery System Self-Assembled from in Situ Dynamic Covalent Reaction of Macrocyclic Disulfides for Cancer Magnetic Resonance Imaging and Chemotherapy.

Supramolecular self-assembly has gained increasing attention to construct multicomponent drug delivery systems for cancer diagnosis and therapy. Despite that these self-assembled nanosystems present surprising properties beyond that of each subcomponent, the spontaneous nature of co-self-assembly causes significant difficulties in control of the synthesis process and consequently leads to unsatisfactory influences in downstream applications. Hence, we utlized an in situ dynamic covalent reaction based on thiol-disulfide exchange to slowly produce disulfide macrocycles, which subsequently triggered the co-self-assembly of an anticancer drug (doxorubicin, DOX) and a magnetic resonance imaging (MRI) contrast agent of ultrasmall iron oxide nanoparticles (IO NPs). It showed concentration regulation of macrocyclic disulfides, DOX, and IO NPs by a dynamic covalent self-assembly (DCS) strategy, resulting in a stable codelivery nanosystem with high drug loading efficiency of 37.36%. More importantly, disulfide macrocycles in the codelivery system could be reduced and broken by glutathione (GSH) in tumor cells, thus leading to disassembly of nanostructures and intellgent release of drugs. These stimuli-responsive performances have been investigated via morphologies and molecular structures, revealing greatly enhanced dual-modal MRI abilities and smart drug release under the trigger of GSH. Moreover, the codelivery system conjugated with a targeting molecule of cyclic Arg-Gly-Asp (cRGD) exhibited significant biocompatibility, MR imaging, and chemotherapeutic anticancer effect in vitro and in vivo. These results indicated that in situ dynamic covalent chemistry enhanced the control over co-self-assembly and paved the way to develop more potential drug delivery systems.

Gold nanorods with iron oxide dual-modal bioprobes in SERS-MRI enable accurate programmed cell death ligand-1 expression detection in triple-negative breast cancer.

The efficiency of immunotherapy for triple-negative breast cancer (TNBC) is relatively low due to the difficulty in accurately detecting immune checkpoints. The detection of TNBC-related programmed cell death ligand-1 (PD-L1) expression is important to guide immunotherapy and improve treatment efficiency. Surface-enhanced Raman spectroscopy (SERS) and magnetic resonance (MR) imaging exhibit great potential for early TNBC diagnosis. SERS, an optical imaging mode, has the advantages of high detection sensitivity, good spatial resolution, and "fingerprint" spectral characteristics; however, the shallow detection penetration of SERS bioprobes limits its application in vivo. MR has the advantages of allowing deep penetration with no radiation; however, its spatial resolution needs to be improved. SERS and MR have complementary imaging features for tumor marker detection. In this study, gold nanorod and ultrasmall iron oxide nanoparticle composites were developed as dual-modal bioprobes for SERS-MRI to detect PD-L1 expression. Anti-PD-L1 (aPD-L1) was utilized to improve the targeting ability and specificity of PD-L1 expression detection. TNBC cells expressing PD-L1 were accurately detected via the SERS imaging mode in vitro, which can image at the single-cell level. In addition, bioprobe accumulation in PD-L1 expression-related tumor-bearing mice was simply and dynamically monitored and analyzed in vivo using MR and SERS. To the best of our knowledge, this is the first time a SERS-MRI dual-modal bioprobe combined with a PD-L1 antibody has been successfully used to detect PD-L1 expression in TNBC. This work paves the way for the design of high-performance bioprobe-based contrast agents for the clinical immunotherapy of TNBC.

Using host-guest interactions at the interface of quantum dots to load drug molecules for biocompatible, safe, and effective chemo-photodynamic therapy against cancer.

Combining photodynamic therapy (PDT) and chemotherapy (CHT) by loading an anti-cancer drug and a photosensitizer (PS) into the same delivery nanosystem has been proposed as an effective approach to achieve synergistic effects for a safe cancer treatment. However, exploring an ideal delivery nanosystem has been challenging, because the noncovalent interactions must be maintained between the multiple components to produce a stable yet responsive nanostructure that takes into account the encapsulation of drug molecules. We addressed this issue by engineering the interfacial interaction between Ag2S quantum dots (QDs) using a pillararene derivative to direct the co-self-assembly of the entire system. The high surface area-to-volume ratio of the Ag2S QDs provided ample hydrophobic space to accommodate the anti-drug molecule doxrubicine. Moreover, Ag2S QDs served as PSs triggered by 808 nm near-infrared (NIR) light and also as carriers for high-efficiency delivery of drug molecules to the tumor site. Drug release experiments showed smart drug release under the acidic microenvironments (pH 5.5) in tumor cells. Additionally, the Ag2S QDs demonstrated outstanding PDT ability under NIR light, as confirmed by extracellular and intracellular reactive oxygen species generation. Significant treatment efficacy of the chemo-photodynamic synergistic therapy for cancer using the co-delivery system was demonstrated via in vitro and in vivo studies. These findings suggest that our system offers intelligent control of CHT and PDT, which will provide a promising strategy for constructing hybrid systems with synergistic effects for advanced applications in biomedicine, catalysis, and optoelectronics.

Amorphous Nitrogen-Doped Carbon Nanocages with Excellent SERS Sensitivity and Stability for Accurate Identification of Tumor Cells.

The surface-enhanced Raman scattering (SERS) bioprobe's strategy for identifying tumor cells always depended on the intensity difference of the Raman signal compared with that of normal cells. Hence, exploring novel SERS nanostructure with excellent spectra stability, a high enhancement factor (EF), and good biocompatibility is a primary premise for boosting SERS signal reliability and accuracy of tumor cells. Here, high SERS EF (5.52 × 106) is acquired by developing novel amorphous nitrogen-doped carbon (NDC) nanocages (NCs), whose EF value was in a leading position among carbon-based SERS substrates. In addition, a uniform SERS signal was obtained on NDC NCs due to homogeneous morphology and size. The delocalized carbon-conjugated systems of graphitic-N, pyrrole-N, and pyridine-N with lone pair electrons increase the electronic density of states and reduce the electron localization function of NDC NCs, thereby promoting the charge transfer process. The electron-donor platform of the NDC NCs facilitates the thermodynamic process of charge transfer, resulting in multimode vibrational coupling in the surface complexes, which greatly amplifies the molecular polarizability. Importantly, the good biocompatibility and signal stability endow these NDC NC SERS bioprobes unique superiority in distinguishing tumor cells, and quantitative recognition of two triple-negative breast cancer cells based on SERS detection mode has been successfully realized.

TiO2-based Surface-Enhanced Raman Scattering bio-probe for efficient circulating tumor cell detection on microfilter.

Circulating tumor cell (CTC) detection as a burgeoning detection strategy can identify the tumor lesion in the early stage, and facilitates the understanding of tumorigenesis, tumor progression, metastasis, and drug-resistance. Herein, we present a novel strategy for in situ isolating and directly detecting CTCs from peripheral blood at single-cell resolution using black TiO2 (B-TiO2)-based Surface-Enhanced Raman Scattering (SERS) bio-probe on a microfilter. CTCs were isolated from blood by microfilter based on the size and deformation difference. The SERS bio-probe was composed of crystal-amorphous core-shell B-TiO2 nanoparticles (NPs), alizarin red (AR) as Raman reporter molecules, and a thin protective layer of NH2-PEG2000-COOH (PEG), which provided sufficient binding sites for target molecule of folic acid (FA). Demonstrated by three cell lines of MCF-7 (folate receptor (FR) positive), A549 and Raw264.7 (FR negative), SERS bio-probe of B-TiO2-AR-PEG-FA could distinguish FR positive CTCs from peripheral blood cells efficiently by targeting FR on CTC membranes and ruling out false positive interference of white blood cells (WBCs) with reliability and specificity. Benefiting by these advantages, this strategy enhanced the detection efficiency and veracity, which reduced the detection time within 1.5 h and make the LOD of detection reduced to 2 cells/mL. These features also facilitated successful CTC detection in several clinical cancer patient bloods which illustrates that the integration of microfluidic isolation and SERS detection may open new paths for liquid biopsy.

A TiO2-based bioprobe enabling excellent SERS activity in the detection of diverse circulating tumor cells.

Circulating tumor cells (CTCs) can be the seeds of tumor metastasis and are closely linked to cancer-related death. Fast and effective detection of CTCs is important for the early diagnosis of cancer and the evaluation of micrometastasis. However, the extreme rarity and heterogeneity of CTCs in peripheral blood make sensitive detection of CTCs a big challenge. In this paper, a TiO2-based surface-enhanced Raman scattering (SERS) bioprobe is reported for the first time with outstanding ultrasensitive specificity, excellent stability of the signal, and good biocompatibility for the detection of CTCs. The TiO2 NPs were encoded with alizarin red (AR) and functionalized with reduced bovine serum protein (rBSA) and folic acid (FA). The limit of detection (LOD) for 4-mercaptobenzoic acid (4-MBA) and AR molecules adsorbed on the TiO2 SERS substrate is 5 × 10-7 M. The designed TiO2-based SERS bioprobe can be effectively utilized in detecting four diverse types of cancer cells in rabbit blood, which shows good sensitivity of the SERS detection technology. Finally, precise targeting of CTCs based on the SERS bioprobe with the function of fluorescence imaging is also confirmed by the fluorescence colouration test. This work offers a novel strategy for CTC detection and the development of non-noble metal semiconductor-based SERS platforms for tumor diagnosis.

Ultrahigh SERS activity of the TiO2@Ag nanostructure leveraged for accurately detecting CTCs in peripheral blood.

Circulating tumor cells (CTCs) usually shed from primary and metastatic tumors serve as an important tumor marker, and easily cause fatal distant metastasis in cancer patients. Accurately and effectively detecting CTCs in a peripheral blood sample is of great significance in early tumor diagnosis, efficacy evaluation, and postoperative condition monitoring. In this work, a TiO2@Ag nanostructure is structured as a SERS substrate, rhodamine 6G (R6G) is used as a Raman signal molecule, the reduced bovine serum protein (rBSA) acts as a protective agent, and folic acid (FA) acts as a target molecule to specifically recognize cancer cells. A TiO2@Ag-based SERS bioprobe is successfully prepared with the feature of ultrahigh sensitivity, good specificity, low toxicity, and high accuracy in CTC detection. The remarkable SERS activity of the TiO2@Ag nanostructure is synergistically contributed by surface plasmon resonance and photon-induced charge transfer mechanism. The limit of detection for rhodamine 6G (R6G) molecules adsorbed on the TiO2@Ag SERS substrate is 5 × 10-14 M, and the corresponding SERS enhancement factor can reach 7.61 × 107. The designed TiO2@Ag-R6G-rBSA-FA SERS bioprobe is effectively utilized in detecting various cancer cells in rabbit blood, and the limit of detection (LOD) for the target cancer cell is 1 cell per mL. Notably, CTCs in peripheral blood of six clinical liver cancer patients are successfully recognized via the TiO2@Ag-based SERS bioprobe. Accurately recognizing CTCs in peripheral blood based on the TiO2@Ag-R6G-rBSA-FA SERS bioprobe is also carefully verified by in situ immunofluorescence staining experiments, which directly supports the CTC detection accuracy of the SERS strategy. These results demonstrate that the TiO2@Ag-based SERS bioprobe has great application potential in early screening and diagnosis of tumors.

Superior Antitumor Efficacy of IFN-α2b-Incorporated Photo-Cross-Linked Hydrogels Combined with T Cell Transfer and Low-Dose Irradiation Against Gastric Cancer.

INTRODUCTION: The exhaustion and poor homing of activated lymphocytes are critical obstacles in adoptive cell immunotherapy for solid tumors. In order to effectively deliver immune cells into tumors, we encapsulated interferon-α2b (IFN-α2b) into macroporous hydrogels as an enhancement factor and utilized low-dose irradiation (LDI) as a tumoral attractor of T cells. METHODS: Hydroxypropyl cellulose hydrogels were prepared by irradiation techniques, and the cross-sectional microstructure was characterized by scanning electron microscopy. The synergistic antitumor mechanism of combination of IFN-α2b and CIK cells was evaluated by detecting the expression of activation marker CD69 on CIK cell surface and IFN-γ production by CIK cells. The in vivo antitumor activity of IFN-α2b-incorporated hydroxypropyl cellulose hydrogels combined with CIK and radiation was evaluated in an MKN-45 xenografted nude mice model. RESULTS: The bioactivity of IFN-α2b was well maintained in ultraviolet-reactive, rapidly cross-linkable hydroxypropyl cellulose hydrogels. In vitro studies demonstrated IFN-α2b-activated T cells, as evidenced by upregulating early activation marker CD69 and secretion inflammatory cytokine IFN-γ. In vivo real-time image showed our hydrogels kept a higher amount of drug delivery at the tumor site for a long time compared with free drug injection. Low-dose irradiation promoted T cell accumulation and infiltration in subcutaneous tumors. Combination of IFN-α2b-loaded hydrogels (Gel-IFN) with T cells and LDI exhibited higher efficacy to eradicate human gastric cancer xenograted tumors with less proliferating cells and more necrotic regions compared with IFN-α2b or T cells alone. DISCUSSION: HPC hydrogels kept the activity of IFN-α2b and stably release of IFN-α2b to stimulate T cells for a long time. At the same time, low-dose radiation recruits T cells into tumors. This innovative integration mode of IFN-α2b-loaded hydrogels and radiotherapy offers a potent strategy to improve the therapeutic outcome of T cell therapy.

Antitumor Activity of Thermosensitive Hydrogels Packaging Gambogic Acid Nanoparticles and Tumor-Penetrating Peptide iRGD Against Gastric Cancer.

INTRODUCTION: Gambogic acid (GA) is proved to have anti-tumor effects on gastric cancer. Due to poor solubility, non-specific biological distribution, toxicity to normal tissues and short half-life, it is hard to be applied into the clinic. To overcome these issues, we developed a thermosensitive and injectable hydrogel composed of hydroxypropyl cellulose, silk fibroin and glycerol, with short gelling time, good compatibility and sustained release, and demonstrated that the hydrogel packaged with gambogic acid nanoparticles (GA-NPs) and tumor-penetrating peptide iRGD could improve the anti-tumor activity. METHODS: The Gelling time and micropore size of the hydrogels were regulated through different concentrations of glycerol. Controlled release characteristics of the hydrogels were evaluated with a real-time near-infrared fluorescence imaging system. Location of nanoparticles from different carriers was traced by confocal laser scanning microscopy. The in vivo antitumor activity of the hydrogels packaging GA-NPs and iRGD was evaluated by investigating tumor volume and tumor size. RESULTS: The thermo-sensitive properties of hydrogels were characterized by 3-4 min, 37°C, when glycerol concentration was 20%. The hydrogels physically packaged with GA-NPs and iRGD showed higher fluorescence intensity than other groups. The in vivo study indicated that the co-administration of GA-NPs and iRGD by hydrogels had higher antitumor activity than the GA-loaded hydrogels and free GA combining with iRGD. Free GA group showed few antitumor effects. Compared with the control group, the body weight in other groups had no obvious change, and the count of leukocytes and hemoglobin was slightly decreased. DISCUSSION: The hydrogel constructed iRGD and GA-NPs exerted an effective anti-tumor effect possibly due to retention effect, local administration and continuous sustained release of iRGD promoting the penetration of nanoparticles into a deep part of tumors. The delivery system showed little systemic toxicity and would provide a promising strategy to improve anti-gastric cancer efficacy.

PD-L1 monoclonal antibody-conjugated nanoparticles enhance drug delivery level and chemotherapy efficacy in gastric cancer cells.

BACKGROUND: Docetaxel (DOC) is widely used as a chemotherapy drug for various tumors, including gastric cancer (GC), but the clinical application of DOC has been limited due to the hydrophobicity of the drug. We aimed to formulate a multifunctional nanoparticle (NP) system to reduce the side effects of the chemotherapy agent, to promote synergistic therapeutic effects, and to achieve targeted delivery of the therapy. METHODS: The polyethylene glycol-poly(ε-caprolactone) NPs (PEG-PCL NPs) were prepared by a ring opening copolymerization technique and were then conjugated with a programmed death-ligand 1 (PD-L1) monoclonal antibody (mAb). The effects of the surface coating on particle size, size distribution, zeta potential, drug encapsulation efficiency, loading capacity, and the drug release kinetics were investigated. By using a panel of PD-L1-expressing human GC cell lines and PD-L1-overexpressing cells, we studied cellular uptake, cytotoxic effects, and cellular apoptosis in the presence of PD-L1 mAb-conjugated NPs. RESULTS: The characterization of the structure and biological functions of DOC-PEG-PCL-mAb NPs was investigated in vitro. X-ray photoelectron spectroscopy validated the presence of the PD-L1 mAbs on the NP surface. The cellular uptake analysis showed that the antibody-conjugated NPs achieved significantly higher cellular uptake. The results of an in vitro cytotoxicity experiment on three GC lines further proved the targeting effects of the antibody conjugation. In addition, we found that the DOC-PEG-PCL-mAb NPs induced cell apoptosis and enhanced G2-M arrest in cancer cells, indicating the inhibition of microtubule synthesis. When compared with the control groups, DOC-PEG-PCL-mAb NPs are more effective in inhibiting PD-L1 expression in GC cells. CONCLUSION: Our results reported here highlight the biological and clinical potential of DOC-PEG-PCL-mAb NPs using PD-L1 mAbs in GC treatment.

Gambogic acid-loaded PEG-PCL nanoparticles act as an effective antitumor agent against gastric cancer.

Poor water solubility and side effects hampered the clinical application of gambogic acid (GA) in cancer therapy. Accordingly, GA-loaded polyethylene glycol-poly(ɛ-caprolactone) (PEG-PCL) nanoparticles (GA-NPs) were developed and administered peritumorally to evaluate their antitumor activity. The particle size, polydispersity index, encapsulation efficiency and loading capacity of GA-NPs were 143.78 ± 0.054 nm, 0.179 ± 0.004, 81.3 ± 2.5% and 14.8 ± 0.6%, respectively. In addition, GA-NPs showed excellent stability, good biocompatibility and sustained release profile. Endocytosis studies in vitro demonstrated that the GA-NPs were effectively taken up by tumor cells in a time-dependent manner. In vivo real-time imaging showed that the nanoparticles effectively accumulated within the tumor tissue after peritumoral administration. The cytotoxicity study revealed that the GA-NPs effectively inhibited the proliferation of gastric cancer cells. In vivo antitumor therapy with peritumoral injection of GA-NPs exhibited superior antitumor activity compared with free GA. Moreover, no toxicity was detected in any treatment group. Histological studies confirmed a lower cell density and a higher number of apoptotic cells in the GA-NPs group compared with the free GA group. Furthermore, the expression level of the cysteine proteases 3 precursor (pro-caspase3), a crucial component of cellular apoptotic pathways, was efficiently reduced in mice treated with GA-NPs. In conclusion, the GA-NPs system provided an efficient drug delivery platform for chemotherapy.

[Evaluation for preparation and anticancer efficacy in vitro of drug-loaded nanoerythrosomes].

The objective of this study is to develop a new-type biodegradable, biocompatible curcumin-loaded nanoerythrosomes (Cur-RBC-NPs) by means of the sonication method. The size of Cur-RBC-NPs was optimized by varying drug loading parameters. The morphology, size distribution, stability, in vitro release pattern, cellular uptake of nanoparticles and in vitro anti-tumor effects were evaluated, respectively. The results showed the prepared Cur-RBC-NPs were nearly uniform spheres, with an average diameter of (245.7 ± 1.3) nm. Encapsulation efficiency (EE) and load efficiency (LE) of Cur-RBC-NPs were 50.65% ± 1.36% and 6.27% ± 0.29%. And the nanoparticles had a good sustained release property. According to the in vitro experiment, Cur-RBC-NPs were effectively taken in by tumor cells, and exhibited a significant anti-tumor effect. In conclusion, the method for preparing Cur-RBC-NPs is convenient, with a good sustained release behavior and anti-tumor efficacy, and so expected to be a new-type nano-drug delivery system in clinical practice.