Ultrasound‐guided percutaneous metal‐organic frameworks based codelivery system of doxorubicin/acetazolamide for hepatocellular carcinoma therapy

Dear Editor, Currently, the enhanced permeability and retention (EPR) effect—one of the main mechanisms through which nanomedicines1 enter solid tumors after intravenous administration—has been mired in controversy due to its variation in different tumors2 and species3 (e.g., insufficient interendothelial gaps in human tumors compared with mouse models). In this study, a zeolitic imidazolate frameworks (ZIF-8) nanocarrier, to facilitate codelivery of doxorubicin (DOX) and acetazolamide (ACE), was prepared using a one-pot process and applied with ultrasound-guided percutaneous intratumoral injection. Rational design of the (DOX+ACE)@ZIF-8 considered the following facts: (a) after single-dose percutaneous

intratumoral injection under ultrasound guidance, the (DOX+ACE)@ZIF-8 tends to directly accumulate at the tumor site and become internalized into tumor cells via endocytosis pathways; (b) ACE, a sulfonamide carbonic anhydrase (CA) inhibitor, currently used as antiepileptic, antiglaucoma, and diuretic agent, 4,5 may have a synergistic effect with DOX and its mechanism may be involved in the ability of inhibiting CA "to acidify the intratumoral environment" 6,7 ; and (c) with intratumoral administration route, the (DOX+ACE)@ZIF-8 shows low systemic toxicity ( Figure 1).
SD rats bearing hepatic in situ tumor models were established to verify the in vivo tumor inhibition capability of (DOX+ACE)@ZIF-8. Figure 4A and B and Video Data S2 display the percutaneous injection procedure involving tumor model establishment. After drug administration, no significant differences in survival ratio were observed from the survival curves ( Figure 4C). Bodyweight of each rat during the entire process was monitored ( Figure 4D). Changes in the tumor volume measured by ultrasound are examined at each scheduled time ( Figure 4E; Figure S5). Furthermore, a series of analyses were carried out including the dissected tumor volume, volume ratio, weight, and H&E staining of tumor-bearing rats sacrificed at the end of the treatment (Figure 4F-J; Figure S6). Notably, our prepared (DOX+ACE)@ZIF-8, combining percutaneous intratumoral injection with ultrasound guidance, achieved significantly better tumor reduction than other groups with intratumoral or intravenous injection.
In vivo fluorescence imaging is a critical technology for the tracking of ZIF-8 based nanocarriers. The drug distribution in isolated organs is shown in Figure S7. After intravenous injection, compared with IR783 solutions, the prolonged systemic circulation of IR783@ZIF-8 was attributed to the EPR effect in rats, reflecting passive tumor-targeted delivery. Interestingly, the rats subjected to percutaneous intratumoral injection of IR820@ZIF-8 exhibited intensive fluorescent signals in the liver, indicating specific drug accumulation of (DOX+ACE)/ZIF-8 at the tumor site.
In summary, a novel strategy by combining (DOX+ACE)@ZIF-8 with ultrasound-guided intratumoral injection, was proposed for HCC therapy. (DOX+ACE)@ZIF-8 was shown to possess uniform cubic morphology, high thermal stability, and pH-responsive release capability. After cellular internalization through the endocytosis pathway, (DOX+ACE)@ZIF-8 specifically released the antitumor drug DOX and sulfonamide CA inhibitor ACE, thereby inhibiting the migration and invasion of Walker 256 cells as well as their cell viability in vitro, which ensured a synergistic antitumor effect. More importantly, compared with drug solutions or intravenous administration, (DOX+ACE)@ZIF-8 exhibited superior antitumor efficacy in vivo with a single dose of percutaneous intratumoral injection under ultrasound guidance. In vivo real-time fluorescence imaging indicated that (DOX+ACE)@ZIF-8 increased drug accumulation at the tumor site. Finally, (DOX+ACE)@ZIF-8 displayed good safety in vivo. This pioneering study proposes a new strategy for the development of nanomedicine with simple processing technology and a non-intravenous administration route, which has potential for further clinical applications in the field of cancer treatment.

C O N F L I C T O F I N T E R E S T
The authors declare no conflict of interest. JZ and WX conceived the experiments; JZ, SZ, LN, ZD,  ZL, and YF performed the in vitro experiments; JZ, WX,  JQ, DS, WM, and CY performed the animal experiments; JZ, DQ, and HY analyzed the data; ZX conceptualized and designed the study, and supervised the project; all authors contributed to the manuscript and the discussion.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.