The role of critical micellization concentration in efficacy and toxicity of supramolecular polymers

Abstract

The inception and development of supramolecular chemistry have provided a vast library of supramolecular structures and materials for improved practice of medicine. In the context of therapeutic delivery, while supramolecular nanostructures offer a wide variety of morphologies as drug carriers for optimized targeting and controlled release, concerns are often raised as to how their morphological stability and structural integrity impact their in vivo performance. After intravenous (i.v.) administration, the intrinsic reversible and dynamic feature of supramolecular assemblies may lead them to dissociate upon plasma dilution to a concentration below their critical micellization concentration (CMC). As such, CMC represents an important characteristic for supramolecular biomaterials design, but its pharmaceutical role remains elusive. Here, we report the design of a series of self-assembling prodrugs (SAPDs) that spontaneously associate in aqueous solution into supramolecular polymers (SPs) with varying CMCs. Two hydrophobic camptothecin (CPT) molecules were conjugated onto oligoethylene-glycol (OEG)-decorated segments with various OEG repeat numbers (2, 4, 6, 8). Our studies show that the lower the CMC, the lower the maximum tolerated dose (MTD) in rodents. When administrated at the same dosage of 10 mg/kg (CPT equivalent), SAPD 1, the one with the lowest CMC, shows the best efficacy in tumor suppression. These observations can be explained by the circulation and dissociation of SAPD SPs and the difference in molecular and supramolecular distribution between excretion and organ uptake. We believe these findings offer important insight into the role of supramolecular stability in determining their therapeutic index and in vivo efficacy.

Keywords: critical micellization concentration; drug delivery; molecular assembly; prodrugs; supramolecular polymers.

Fig. 1.

Schematic illustration of the design and self-assembly of self-assembling prodrugs (SAPDs). (A) Chemical structure of the designed SAPDs. (B) Cartoon of SAPD platform. Two hydrophobic camptothecin (CPT) molecules (yellow) were conjugated with four different oligoethylene-glycol (OEG)-decorated hydrophilic auxiliaries (blue) through the biodegradable etcSS linker (black) to create SAPDs 1 to 4, respectively. (C) Illustration of self-assembly of SAPD into supramolecular polymer (SP).

Fig. 2.

Supramolecular polymers (SPs) formed by SAPDs in water. Representative cryo-TEM of supramolecular assemblies of SAPD 1 (A), SAPD 2 (B), SAPD 3 (C), and SAPD 4 (D). TEM images reveal that all of the prodrugs self-assembled into 1D structures. All concentrations: 2 mM.

Fig. 3.

CMC, stability, and drug release studies of SAPDs. (A) CMC measurement of SAPDs using a Nile Red method. CMCs of SAPDs 1 and 2 are estimated to be 2.7 and 10.1 μM, respectively. CMCs of SAPDs 3 and 4 exceed 200 μM, and the exact values cannot be directly determined here. (B) CD spectra of SAPDs at 200 μM in water. SAPD 1 shows very strong absorptions attributed to CPT chromophore interactions and intermolecular hydrogen bonding. SAPD 2 shows a similar pattern with largely reduced intensities. The lack of typical hydrogen-bonding interactions and characteristic CPT absorptions in SAPDs 3 and 4 indicates that they may not form 1D nanostructures at the concentration of 200 μM. The chromophore absorptions can be ascribed to intramolecular CPT interactions within a single prodrug. (C) CD spectra of SAPDs at 200 μM in 10% rat plasma. No apparent difference in the absorptions of SAPDs 1 and 2 were observed compared with those in water, while slight changes can be seen in the cases of SAPDs 3 and 4. (D) Plots of absorption of SAPD 1 and SAPD 2 assemblies at 389 nm in the time- and concentration-dependent CD measurement. Drug release plots of SAPDs at 200 μM in PBS (E) and rat plasma (G) with 10 mM GSH. Cumulative drug degradation plots of SAPDs at 200 μM in PBS (F) and rat plasma (H) without GSH. n = 3 for all drug release studies.

Fig. 4.

In vitro cell cytotoxicity of SAPDs against HT-29 colorectal adenocarcinoma cells (A) and HCT-116 colorectal carcinoma cells (B), with both free CPT and irinotecan as controls (72-h incubation, n = 3).

Fig. 5.

In vivo antitumor efficacy and circulation study of SAPDs at the same dose (10 mg/kg, CPT equivalent). SAPDs were i.v. injected q4dx4 (black arrows) at days 1, 5, 9, and 13 at a dose of 10 mg/kg mice (n = 6). Blank PBS group and free CPT (i.p.) at a dose of 9 mg/kg (q4dx4) at days 1, 5, 9, and 13 were taken as controls (n = 5); however, administration of CPT resulted in death of all five mice after the second dose. Irinotecan (i.p.) at a dose of 100 mg/kg (qwkx3, red arrows) at days 1, 8, and 15 was another control (n = 5). Tumor volume (A), body weight (B), and cumulative survival (C) plots of mice. Loss of mice is a result of treatment-related death or killing after predetermined end point was reached. All of the data are presented as mean ± SD and analyzed by one-way ANOVA (Fisher; 0.01 < *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Real-time concentration of total CPT (D) and bounded CPT in SAPDs (E) in the circulation study on SD rats (n = 3) at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 12 h. (F) The ratio of bounded CPT in SAPDs to total CPT within 1 h after injection.

Fig. 6.

In vivo antitumor efficacy of SAPDs at their respective estimated MTDs. SAPDs were i.v. injected q4dx3 (black arrows) on days 1, 5, and 9 at doses of 12 mg/kg (CPT equivalent) for SAPD 1, and 36 mg/kg for all other three SAPDs (n = 5). Blank PBS group (n = 5) was taken as a control, and irinotecan (i.p.) at a dose of 100 mg/kg (qwkx3, red arrows) on days 1, 8, and 15 was another control (n = 5). Tumor volume (A), body weight (B), and cumulative survival (C) plots of mice. Slight decrease of body weight was observed in all treated groups with one treatment-related death in the SAPD 2 group. All of the data are presented as mean ± SD and analyzed by one-way ANOVA (Fisher; 0.01 < *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Scheme 1.

Illustration of the circulation fate of a supramolecular polymer (SP) after entering into the circulation. SP (1) may dissociate into fragments/monomers in the plasma upon dilution, and the dissociation kinetics is mostly dictated by its CMC. SP has the tendency to accumulate more in the tumor (2) and major organs (3) (liver, spleen, kidney, lung, and heart), while fragments/monomers (4) tend to undergo a rapid renal clearance. Thus, the lower the CMC of a SP, the lower the percentage of fragments and monomers, leading to reduced excretion and enhanced tumor (improved efficacy) and healthy organ uptake (increased toxicity).