Background: The application of combination therapy for cancer treatment is limited due to poor tumor-specific drug delivery and the abscopal effect. Methods: Here, PD-L1- and CD44-responsive multifunctional nanoparticles were developed using a polymer complex of polyethyleneimine and oleic acid (PEI-OA) and loaded with two chemotherapeutic drugs (paclitaxel and chloroquine), an antigen (ovalbumin), an immunopotentiator (CpG), and an immune checkpoint inhibitor (anti-PD-L1 antibody). Results: PEI-OA greatly improved the drug loading capacity and encapsulation efficiency of the nanoplatform, while the anti-PD-L1 antibody significantly increased its cellular uptake compared to other treatment formulations. Pharmacodynamic experiments confirmed that the anti-PD-L1 antibody can strongly inhibit primary breast cancer and increase levels of CD4+ and CD8+ T cell at the tumor site. In addition, chloroquine reversed the “immune-cold” environment and improved the anti-tumor effect of both chemotherapeutics and immune checkpoint inhibitors, while it induced strong immune memory and prevented lung metastasis. Conclusions: Our strategy serves as a promising approach to the rational design of nanodelivery systems for simultaneous active targeting, autophagy inhibition, and chemotherapy that can be combined with immune-checkpoint inhibitors for enhanced breast cancer treatment.

Parvovirus B19 (B19V) is pathogenic to humans and causes various human diseases. However, no antiviral agents or vaccines currently exist for the treatment or prevention of B19V infection. Therefore, developing sensitive and specific methods for B19V infection diagnosis is essential for accurate diagnoses. Previously, a Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas12a (cpf1)-based electrochemical biosensor (E-CRISPR) with a picomole sensitivity for B19V detection was established. Herein, we set up a novel nucleic acid detection system based on Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection, targeting the nonstructural protein 1 (NS1) region of the B19V viral genome (abbreviated B19-NS1 PAND). Benefiting from independent protospacer adjacent motif (PAM) sequences, PfAgo can recognize their target with guide DNA (gDNA) that is easy to design and synthesize at a low cost. In contrast to E-CRISPR, without preamplification with Polymerase Chain Reaction (PCR), the Minimum Detectable Concentration (MDC) of three guide- or single guide-mediated B19-NS1 PAND was about 4 nM, approximately 6-fold more than E-CRISPR. However, when introducing an amplification step, the MDC can be dramatically decreased to the aM level (54 aM). In addition, the diagnostic results from clinical samples with B19-NS1 PAND revealed 100% consistency with PCR assays and subsequent Sanger sequencing tests, which may assist in molecular testing for clinical diagnosis and epidemiological investigations of B19V.

The main obstacles to the clinical application of the CRISPR/Cas9 system are off-target effects and low delivery efficiency. There is an urgent need to develop new delivery strategies and technologies. Three types of in situ injectable hydrogels with different electrical properties were created to find the most secure and efficient sustained-release drug delivery system. After in vitro and in vivo comparisons, we found that the positively charged hydrogels had higher cellular uptake, stronger gene editing efficiency, greater cytotoxicity, longer tumor accumulation, and better anti-tumor efficacy than negatively charged and neutral hydrogels. We designed single guide RNA targeting the Y-box binding protein 1 (YB-1) gene and then used it to create a ribonucleoprotein complex with Cas9 protein. Doxorubicin was co-encapsulated into this positively charged hydrogel to create a co-delivery system. By knocking down YB-1, the expression of YB-1 was reduced, inhibiting the growth and migration of melanoma cells. The strategy of combining YB-1 gene editing and intratumoral injection enhanced the therapeutic effect of doxorubicin while reducing side effects.

The CRISPR/Cas9 system-based gene therapy can fundamentally address the issues of cancer occurrence, development, progression, and metastasis. However, the lack of targeting and effectiveness hinders gene therapy from entering clinical application. Herein, a somatostatin receptor-targeted polymeric nanoplatform is developed for the delivery of a PD-L1-targeted CRISPR/Cas9 system and synergistic treatment of hepatocellular carcinoma. This nanoplatform can effectively incorporate the CRISPR/Cas9 system and the chemotherapeutic drug paclitaxel to simultaneously address the biological safety and packaging capacity issues of viral vectors. After the octreotide-modified polymer (LNA-PEG-OCT) guided the nanoparticle into hepatoma carcinoma cells, the nanoparticle protected the CRISPR/Cas9 ribonucleoprotein complex (RNP) and achieved lysosomal escape. Then, the RNP reached the target gene (PD-L1) under the guidance of the single guide RNA (sgRNA) in the RNP. The PD-L1 gene editing efficiency reached up to 55.8% for HepG2 cells in vitro and 46.0% for tumor tissues in vivo, leading to effective suppression of PD-L1 protein expression. Substantial inhibition of hepatocellular carcinoma cell proliferation and further 79.45% growth repression against subcutaneous xenograft tumors were achieved. Overall, this somatostatin receptor-targeted polymeric nanoplatform system not only provides a promising nanocarrier for CRISPR/Cas9 system delivery, but also expands the potential of combining gene editing and chemotherapy.