Transferrin-binding peptide functionalized polymersomes mediate targeted doxorubicin delivery to colorectal cancer in vivo
Graphical abstract
Transferrin (Tf)-binding peptide CGGGHKYLRW functionalized polymersomes selectively and stably bind Tf and subsequently mediate targeted delivery of doxorubicin to TfR over-expressing HCT-116 colorectal cancer cells in vivo.
Introduction
Targeted nanomedicines are considered as a future treatment modality for cancers [[1], [2], [3], [4]]. To accomplish targeted delivery, researchers have decorated nanomedicines with different ligands ranging from peptides [5,6], antibodies [7] and antibody fragments [8], glycoproteins [[9], [10], [11]], to folic acid [12,13]. In spite of intensive investigations, few actively targeted nanomedicines have come to the stage of clinical translation [[14], [15], [16]]. Notably, several targeted nano-formulations homing to transferrin receptor (TfR) have been approved for clinical trials [[17], [18], [19]]. TfR over-expresses on highly proliferative cancer cells [10,12,20]. Transferrin (Tf) and single-chain antibody fragment (ScFv) against TfR have been selected as ligands for TfR targeting [[21], [22], [23], [24], [25]]. For example, Davis et al. described that Tf-conjugated, cyclodextrin polymer-based nanoparticles exhibited an enhanced transfection of K562 leukemia cells as compared to the non-targeted control [26], and evidence of RNAi in a phase I clinical trial targeted delivery of siRNA to patients with solid tumor [27]. Chang et al. reported that liposomes modified with ScFv could mediate targeted delivery of wild-type p53 gene to metastatic pancreatic tumor. The results from phase I clinical trial revealed the accumulation of p53 gene in advanced solid tumor in patients, low adverse effects, and stablized disease [28]. However, the conjugation of large ligands like Tf and ScFv to nanomedicines via post-modification may pose significant production challenges. The post-modification with large ligands may encounter issues like poor control over conjugation site and efficacy, and difficulty in purification and in scaling up production.
In contrast to large glycoproteins and antibody fragments, peptides with a short sequence and easy handling enable functionalization of nanomedicines via pre-modification [29]. Various peptides have been screened as antibody alternatives for targeted tumor therapy in the past years [[30], [31], [32], [33], [34], [35]]. Nevertheless, to date, only BIND-014, a docetaxel nanoformulation decorated with prostate-specific membrane antigen-targeting peptide, has reached clinical assessments [36]. The slow development of peptide-targeted nanomedicines is likely due to the fact that peptides are not as specific and effective as antibodies for in vivo targeting. Interestingly, Signore et al. reported that CGGGHKYLRW as a Tf-binding peptide (TBP) showed a high specificity and affinity to Tf [37]. TBP following plasma incubation could promote TfR-mediated cellular uptake of gold nanoparticles.
Here, we report for the first time that Tf-binding peptide-functionalized polymersomes (TBP-Ps) loaded with doxorubicin hydrochloride (TBP-Ps-Dox) following Tf binding could mediate targeted Dox delivery to TfR over-expressing HCT-116 colorectal cancer cells in vitro and in vivo (Scheme 1). Several reports demonstrated that colorectal cancers over-express TfR [38,39]. We previously reported that the disulfide-crosslinked polymersome is a promising substitute to liposome for Dox delivery [[40], [41], [42], [43]]. Interestingly, TBP-Ps-Dox following Tf binding (Tf@TBP-Ps-Dox) revealed greatly enhanced cellular uptake and antitumor effect in HCT-116 cells over Ps-Dox. The pharmacokinetics and anti-tumor therapy experiments revealed that Tf@TBP-Ps-Dox exhibited a long circulation time and a considerably improved inhibition of HCT-116 tumor as compared to Ps-Dox. Surface functionalization of the nanoparticles with Tf-binding peptide thus provides an appealing strategy to fabricate nanomedicines targeting to TfR over-expressing malignancies.
Section snippets
Synthesis of TBP-PEG-P(TMC-DTC)
Poly(ethylene glycol)-b-poly(trimethylene carbonate-co-dithiolane trimethylene carbonate) (mPEG-P(TMC-DTC)) and maleimide functionalized copolymer Mal-PEG-P(TMC-DTC) were produced according to our previous report (Table S1) [40]. Mal-PEG-P(TMC-DTC) (200 mg, 8.2 μmol) was added under stirring to 1.5 mL TBP (CGGGHKYLRW, 19.2 mg, 16.4 μmol) solution in N, N-dimethlyformamide (DMF). The reaction proceeded at 37 °C for 24 h, followed by intensive dialysis (MWCO 3500) against 100 mL DMF (× 3) and
Formation and characterization of TBP-Ps-Dox and Tf@TBP-Ps-Dox
TBP-Ps-Dox with three different TBP molar surface densities were obtained from co-assembly of PEG-P(TMC-DTC) and 8.6, 17.2 and 25.8 mol% TBP-PEG-P(TMC-DTC) (referring to total copolymers), followed by Dox·HCl loading using a pH gradient method (Scheme 1A). To make TBP preferentially located at the outer surface of polymersomes facilitating the Tf binding, the PEG in TBP-PEG-P(TMC-DTC) (Mn = 7.5 kg/mol) was designed longer than that in PEG-P(TMC-DTC) (Mn = 5.0 kg/mol) (Table S1). The dynamic
Conclusion
We have demonstrated that disulfide-crosslinked polymersomes functionalized with a transferrin-binding peptide (TBP-Ps) can selectively and stably bind transferrin and subsequently mediate targeted Dox delivery to TfR over-expressing HCT-116 colorectal cancer cells in vitro and in vivo, leading to enhanced tumor suppression and reduced off-target side effects. To the best of our knowledge, this is the first report on the preparation of transferrin-functionalized nanomedicines by selective
Acknowledgements
This work is supported by research grants from the National Natural Science Foundation of China (NSFC 51633005, 51761135117, 51773146, 51861145310, 51561135010).
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