Acute myeloid leukemia (AML) is a malignancy characterized by the clonal proliferation of myeloid hematopoietic progenitor or stem cells, primarily affecting adults with a relatively high incidence and poor prognosis, with a 5-year survival rate of only 25% to 40%. Although the "7 + 3" induction chemotherapy regimen remains the standard treatment for AML, its nonselective cytotoxic mechanism often results in severe myelosuppression and organ toxicity, thus limiting long-term efficacy. AML exhibits considerable biological heterogeneity, with multiple driver gene mutations such as FLT3-ITD, CEBPA, IDH1/2, and DNMT3A identified as closely associated with its progression. Targeted therapies developed based on these mutations show promising therapeutic potential. However, approximately 30% to 40% of AML patients lack clearly defined druggable targets, especially in monocytoid subtypes such as M4 and M5, which are characterized by high aggressiveness and poor prognosis. "Therefore, developing precise therapeutic strategies involving multi-mechanistic synergies is a key research direction for advancing AML treatment. " said the author Haiyin Yang, a researcher at Beijing Institute of Technology, "In this study, we introduce a carrier-free "3-in-1" peptide–daunorubicin–siRNA (PDR) nanoassembly designed for targeted therapy of AML, offering a new solution."
The PDR nanoassembly consists of three functional components: 1) a tumor-suppressing peptide (CPP44-p16MIS-FFY-4R), 2) a pH-sensitive daunorubicin (DNR) prodrug (PEG-Hyd-DNR), and 3) siRNA targeting the LILRB4 gene (siLILRB4). CPP44 is a novel cell-penetrating peptide that enables efficient endocytosis and selective delivery to AML cells. p16MIS mimics the functional domain of the p16 protein, inhibiting CDK4/6 activity to induce G1-phase cell cycle arrest. FFY is a self-assembling tripeptide that drives the core assembly of the DNR prodrug via π–π stacking. The 4 arginine residues (4R) bind siRNA through electrostatic interactions and facilitate endosomal escape. The DNR prodrug is formed by linking DNR to PEG-2000 through a hydrazone bond, which is pH-responsive. This bond is cleaved in the acidic environment of the endosome, releasing the DNR drug. siLILRB4 is a small interfering RNA that targets the LILRB4 gene to relieve immune suppression and enhance anti-leukemic immune responses. PDR nnanoassembly were prepared by nano precipitation method. Firstly, DNR is linked to PEG-2000 via pH sensitive hydrolysis amide bonds to form DNR prodrug (PEG Hyd DNR). The prodrug can effectively hydrolyze and release DNR drugs in an acidic internal environment. Then mix the DNR prodrug, functionalized peptide, and siRNA in a specific molar ratio in an aqueous solution, stir for a certain period of time, and form PDR nanoparticles. Finally, the solvent was removed by dialysis to obtain PDR nanoparticles.
The authors conducted a comprehensive characterization and efficacy evaluation of PDR. Through dynamic light scattering (DLS) and transmission electron microscopy (TEM), PDR nanoparticles were shown to have a spherical structure, with a particle size of approximately 150 nm and good dispersion (PDI = 0.079), indicating good uniformity and stability. The study then assessed the cellular uptake efficiency and gene silencing effects of PDR in AML cells. The results demonstrated that the cellular uptake efficiency of PDR significantly increased with higher proportions of DNR prodrug. Flow cytometry (FACS) confirmed that PDR efficiently entered AML cells and achieved effective gene silencing. Real-time qPCR analysis further showed that PDR significantly inhibited the expression of the LILRB4 gene, demonstrating its expected targeted therapeutic effect. Regarding pH-sensitive release, PDR exhibited rapid siRNA release in acidic environments (pH 5.5), with a release rate of up to 70%, while in physiological pH conditions (pH 7.4), the release rate was slower, with only 20% released within 24 hours. This suggests that PDR can rapidly release drugs in the endosomal environment, demonstrating good endosomal escape properties. In terms of anti-leukemia efficacy, PDR combination therapy significantly enhanced cytotoxicity in AML cells, inhibited cell proliferation, and promoted apoptosis. Compared to DNR or siLILRB4 alone, the combination treatment with PDR exhibited stronger cytotoxicity and anti-leukemia effects. In vivo mouse model experiments showed that PDR nanoparticles exhibited excellent targeting and effectively inhibited tumor growth. Compared to conventional chemotherapy drugs, PDR significantly improved mouse survival rates. Finally, safety evaluation of PDR showed no significant toxic reactions in vivo. Histological analysis showed no organ damage, and PDR demonstrated good biodistribution and biocompatibility in vivo. These results indicate that PDR nanoassemblies have good therapeutic efficacy and safety, and hold promise as a potential therapeutic strategy for AML.
In conclusion, this study successfully developed a self-assembled, carrier-free nanotherapeutic system—PDR—composed of a multifunctional peptide, a pH-responsive DNR prodrug, and siRNA targeting LILRB4. This platform integrates targeted delivery, chemotherapy, and gene silencing to achieve multi-mechanistic anti-leukemic effects. The peptide component not only enables selective recognition and internalization into AML cells via the CPP44 domain but also delivers the tumor-suppressive p16MIS sequence, which induces phase arrest and apoptosis by disrupting cell cycle progression. Upon endocytosis, the hydrazone bond in the DNR prodrug is cleaved in the acidic endosomal environment, facilitating both endosomal escape and intracellular drug release. Concurrently, siRNA-mediated silencing of LILRB4 reduces leukemic cell infiltration into major organs and enhances T cell maturation, contributing to an immune-mediated anti-leukemic response. Collectively, the PDR nanodrug demonstrates potent anti-AML efficacy by combining cell cycle interference, chemotherapeutic cytotoxicity, and immunomodulation, demonstrating its strong potential for clinical translation in AML therapy. "The PDR nanomedicine demonstrates strong anti AML efficacy by combining cell cycle disruption, chemotherapy cytotoxicity, and immune regulation, and has strong clinical translational potential in AML treatment." said Haiyin Yang.
Authors of the paper include Haiyin Yang, Xi Yu, Zhitong Guo, Songxuan Shi, Jie Wang, Shuai Guo, Bo Hu, Meihong Chai, Zhuoran Wang, Stefan Barth, Kelong Fan, Huining He, Mengjie Zhang, and Yuanyu Huang.
This work was supported by the Beijing Nova Program (Interdisciplinary Cooperation Project) from Beijing Municipal Science & Technology Commission (20220484207), the National Key Research & Development Program of China (2021YFE0106900), the National Natural Science Foundation of China (32171394 and 32401187), the Postdoctoral Science Foundation of China (2023M740258), the Postdoctoral Fellowship Program of CPSF (GZC20233397), the Fundamental Research Funds for the Central Universities (2022CX01013), the Natural Science Basic Research Program of Shaanxi (2024JCYBQN0929), and Xi'an Science and Technology Plan Project (23YXYJ0067).
The paper, "Carrier-Free Peptide–Daunorubicin–Small Interfering RNA Nanoassembly for Targeted Therapy of Acute Myeloid Leukemia" was published in the journal Cyborg and Bionic Systems on Nov. 5, 2025, at DOI: 10.34133/cbsystems.0436.
