2.1. Design, Synthesis, and Optimization of CD Derivatives
β-CD was selected as the core material to build organic renal-clearable NCs because of its several advantages, including biocompatibility and biodegradability, lack of pharmacological activity and immunogenicity, and a polyfunctional structure exhibiting hydrophobic cavity for host–guest interactions with various compounds, such as drugs and imaging probes42. Various charged moieties were introduced to the side chains of β-CD to reduce off-target accumulation, as the surface charge has a significant impact on the in vivo fate of NCs, especially those of ultrasmall sizes19,20,21,27,28. Sulfobetaine was adopted as a zwitterionic moiety because it can resist non-specific protein adsorption more efficiently than other amino acids43. Based on the general understanding of the structure–antifouling efficiency relationship of zwitterionic molecules44, we prepared two types of sulfobetaine (compounds 2 and 4) with different numbers of methylene groups between quaternary ammonium and carboxylic acid (Figure S1). Succinic anhydride, betaine hydrochloride, and glycolic acid were used for the preparation of the negative, positive, and uncharged CD derivatives, respectively. Phenylboronic acid (PBA) was chosen as a tumor-targeting probe because it can recognize sialyl epitopes that are overexpressed in CRC cells45. The neutral charge of PBA at physiological pH was also suitable for minimizing the unintended charge effects by targeting ligands on the entire NC structure46,47. Glutamic acid was used as a tri-arm linker to tether both the charged moiety and the targeting ligand to the CD core.
Twenty CD derivatives (CD 1–20) were designed by modifying the charged moiety linked to the amine group of the glutamic acid linker (P1) and spacers between cyclodextrin and glutamic acid (P2), and glutamic acid and PBA (P3) (Fig. 1A). Synthesis was performed through the following three main steps: (1) synthesis of PBA–P3 (zero-length or PEG4)–glutamic acid (compounds 6 and 10), (2) conjugation of (1) and CD–P2 (zero-length or spacers) (compounds 12, 19, 20, 28, and 30), and (3) conjugation of (2) and P1 (charged moieties). Each module (A–F) was conjugated via amide coupling, and the detailed synthetic schemes are described in the Supporting Information (Section S1.2 and Figure S2). Successful syntheses of the intermediates were confirmed using proton nuclear magnetic resonance (1H-NMR) spectroscopy and mass spectrometry (Figures S3–S7). The degrees of conjugation of PBA or spacers (PEGn and caproic acid) to CD intermediates were calculated by comparing integration values of anomeric protons of CD (~ 4.8 ppm) and distinct signals of the conjugated compounds (8.0 and 1.3 ppm for PEGn; 1.2–1.5 ppm for caproic acid; and 9.8, 6.8, and 1.3 ppm for PBA). The introduction of PBA was further confirmed via the alizarin red S test, wherein the CD intermediates (compounds 11, 17, 18, 27, and 29) were nearly heptaconjugated (Figure S8). In synthesis step (3), excess charged moieties were used to convert all the primary amines on the glutamic acid linker. The fluorescamine assay revealed that more than 98.8% of the primary amines were consumed within 48 h, indicating the complete conjugation of the charged moieties (Figure S9). This result was further supported by the 1H-NMR spectra of the final products, CD 16–20 (Figure S10).
The foremost requirement for intravenous injections is colloidal stability in the bloodstream. It should be noted that CD derivatives may become more vulnerable to aggregation upon the formation of inclusion complexes with hydrophobic guest molecules. Thus, we screened out CD derivatives with poor colloidal stability in phosphate-buffered saline (PBS; high ionic strength condition) or fetal bovine serum (FBS; bloodstream condition) based on turbidimetric analysis by measuring absorbance at 600 nm (Figure S11A, summarized in Fig. 1B). CD derivatives with no spacers (i.e., zero length) at P2 and P3 (CD 1, 2, 4, and 5) exhibited precipitation when dispersed in PBS and FBS. The introduction of a short PEG4 spacer at P2 was insufficient to secure colloidal stability in PBS (CD 6, 7, 9, and 10); CD 8, 9, and 10 were readily dissolved in FBS, while CD 6 and 7 showed severe aggregation. These results can be attributed to the strong inter- and intramolecular interactions of the zwitterionic moieties rather than the inherent hydrophobicity of the CD molecules48. Accordingly, CD derivatives with longer spacers (PEG8) at P2 (CD 11, 13, 14, and 15) were dissolved in PBS and FBS. Interestingly, however, the A2 module (compound 4)-conjugated CD derivative (CD 12) precipitated in both media, even with the PEG8 spacer. Thus, the A1 module (compound 2) was selected as the zwitterionic functional group for further study. Next, the length of the PEG spacer was extended to further improve the colloidal stability. For CD 16, PBA and A1 were directly conjugated to glutamic acid and a long PEG12-caproic acid spacer was inserted at P2. In contrast, CD 17–20 were prepared by inserting a PEG8-caproic acid spacer at P2 and the PEG4 spacer at P3, hypothesizing that nearby sulfobetaine could hinder the sialic acid-targeting ability of PBA. As expected, CD 16–20 were dissolved clearly in both media and exhibited no significant turbidity changes for 48 h, suggesting high colloidal stability after intravenous administration (Figure S11B). Moreover, dynamic light scattering (DLS) analysis revealed that the hydrodynamic diameters (HDs) of CD 16–20 were of renal-clearable size (5–6.6 nm), and the zeta potential of each CD derivative corresponded to the decorated charged moieties, supporting the successful synthesis of CD derivatives (Fig. 1C).
2.2. Biodistribution of CD Derivatives
Adamantyl sulfocyanine 7 (ACy7) was synthesized as a fluorescence probe for the near-infrared fluorescence (NIRF) imaging-assisted biodistribution study of CD derivatives (Figure S12A). Given its high affinity to the hydrophobic cavity of CD, ACy7 was successfully loaded onto CD 16–20 by inducing host-guest interactions under aqueous conditions49,50, resulting in stable CD/ACy7 inclusion complexes. Although CD 11 and 14 had decent colloidal stability (Fig. 1B), gradual precipitation was observed in the presence of ACy7 (Figure S12B), discouraging further evaluation of these two compounds.
Next, free ACy7 and CD 16–20/ACy7 inclusion complexes were intravenously administered to a CRC mouse model, and ex vivo imaging was performed 24 h post-injection (Fig. 1D). In this study, HT-29 (human colorectal adenocarcinoma) was utilized because this cell line represents the traits of human CRC, including the high expression of sialyl Lewis A, a well-known target of PBA51. Compared to free Acy7, which showed non-selective organ distribution, CD 16/ACy7 (zwitterionic) exhibited significantly reduced lung and liver accumulation with increased kidney selectivity. These results imply that zwitterionic CD derivatives may reduce the off-target distribution or retention of cargo molecules and facilitate renal excretion. A similar phenomenon was observed in the case of CD 17/ACy7, another zwitterionic inclusion complex, wherein the average fluorescence intensity values of ACy7 in normal tissues were almost at the same levels as those of CD 16/ACy7. However, the tumor distribution efficiency of CD 17/ACy7 was 2.02-fold higher than that of CD 16/ACy7 (p < 0.001) (Fig. 1E), even with similar physicochemical properties (Fig. 1C). This result could be attributed to the insertion of the PEG4 spacer between the PBA moiety and glutamic acid linker, reducing the possible interference of sulfobetaine in the PBA–sialic acid interaction (Fig. 1F). Although CD 18/ACy7, a negatively charged inclusion complex, displayed an overall high tumor-to-background ratio (TBR) comparable to that of CD 17/ACy7, the tumor distribution efficiency was 1.46-fold lower (p < 0.01) (Fig. 1E). Conversely, CD 19/ACy7 (positively charged) and CD 20/ACy7 (neutral) showed comparable tumor distribution efficiencies to CD 17/ACy7, but their TBR values were approximately 2-fold lower in all organs (Fig. 1G). Based on high TBR and tumor distribution efficiency, CD 17, hepatkis-(6-deoxy-6-((phenylboronic acid-tetraethyleneglycol-l-glutamic acid Nα-sulfobetaine)-octaethyleneglycol-caproamide))-β-cyclodextrin (PBA-(ZW)-CD), was selected as an optimized renal clearable CD derivative for further investigations.
2.3. Tumor Targetability, Pharmacokinetics, and Urinary Excretion of PBA-(ZW)-CD/ACy7
The structural components of PBA–(ZW)–CD are shown in Fig. 2A. The caproic acid linker enhanced hydrophobic interactions with guest molecules52,53, and the PEG8 spacer improved colloidal stability. Sulfobetaine, a zwitterionic functional group, reduces non-specific tissue binding54,55. The PEG4 spacer between glutamic acid and PBA helped preserve the targeting ability of PBA. These features may translate into improved in vivo distribution profiles (Fig. 2B). Thus, we hypothesized that PBA-(ZW)-CD/ACy7 could be rapidly distributed throughout the body and selectively retained in tumor tissues via PBA–sialic acid interactions. In addition, the zwitterionic sulfobetaine moiety could reduce non-specific interactions between PBA-(ZW)-CD and normal tissues; thus, the clearance of ACy7 may be accelerated via the renal route, further enhancing tumor selectivity.
Prior to verifying the targetability of PBA-(ZW)-CD in vivo, enhancement in the cellular uptake of PBA-(ZW)-CD/ACy7 via PBA–sialic acid interaction was confirmed in HT-29 cells by fluorescence microscopy (Figure S13A). Notably, the cellular uptake efficiency of PBA-(ZW)-CD/ACy7 was compromised in the presence of free sialic acid, a competitive inhibitor, suggesting the involvement of PBA–sialic acid interactions in the cellular uptake of PBA-(ZW)-CD. Similarly, flow cytometry analyses were performed on HT-29 cells treated with the inclusion complex of adamantyl sulfocyanine 5 (Figure S12A; Figure S13B), and the results corresponded well with the fluorescence microscopy data, supporting the use of PBA as an HT-29 targeting moiety.
The tumor targeting ability of PBA-(ZW)-CD/ACy7 was investigated in HT-29 tumor-xenograft mice using real-time NIRF imaging (Fig. 2C). In the early phase (~ 2 h), intravenously administered ACy7 and PBA-(ZW)-CD/ACy7 were both distributed throughout the body with no tumor selectivity. However, after rapid renal excretion of PBA-(ZW)-CD, which was verified in a preliminary study with sulfocyanine 7-conjugated PBA-(ZW)-CD (i.e., via covalent bond) (Figure S14), the PBA-(ZW)-CD/ACy7 inclusion complex-treated mice exhibited distinct fluorescence signals in the tumor and kidney regions in whole-body imaging (Fig. 2C). Furthermore, the TBR of PBA-(ZW)-CD/ACy7 exceeded 2, the standard cut-off value for tumor detection56, at 8 h post-injection and continuously increased for the next 16 h, whereas free ACy7 showed TBR values less than 2 throughout the experimental period (Fig. 2D). The high TBR of PBA-(ZW)-CD/ACy7 can be attributed to prolonged tumor retention and accelerated elimination from normal tissues. From a clinical perspective, determining the tumor margin is of paramount importance for the accurate surgical resection of CRC, especially in laparoscopic or robot-assisted operations56,57,58. To evaluate the applicability of PBA-(ZW)-CD/ACy7 in image-guided surgery, PBA-(ZW)-CD/ACy7 was injected into the tail vein of an orthotopic CRC model, which was prepared by subserosal inoculation of HT-29 cells in the cecum after laparotomy. At 24 h post-injection, PBA-(ZW)-CD/ACy7 showed high TBR values of approximately 4 against adjacent normal tissues, including the small intestine, colon, and muscle, providing accurate tumor margins for resection (Fig. 2E and 2F).
The pharmacokinetic properties of PBA-(ZW)-CD/ACy7 were investigated via time-course blood sampling and ACy7 quantification (Figure S15). Exhibiting biphasic decay, free ACy7 and PBA-(ZW)-CD/ACy7 showed similar distribution half-lives (t1/2α; 4.2 and 3.0 min, respectively). This result suggests that PBA-(ZW)-CD/ACy7 can be distributed to the whole body as quickly as free ACy7, as observed in the in vivo imaging study (Fig. 2C). However, in the elimination phase, PBA-(ZW)-CD/ACy7 had a significantly shorter elimination half-life (t1/2β; 52.5 min) than free ACy7 (92.5 min). Considering comparable exposure, the reduced t1/2β value could result from the lower volume of distribution (Vd) of the inclusion complex (5.6 mL) compared to that of the free dye (7.6 mL), which also implies reduced non-specific binding of ACy7 in normal tissues. Although the total body clearance values of free dye (0.08 mL min–1) and inclusion complex (0.10 mL min–1) were almost the same, the cumulative urinary excretion of PBA-(ZW)-CD/ACy7 was 2.3-fold higher than that of free ACy7, which suggests that PBA-(ZW)-CD may facilitate the renal excretion of cargo molecules (Fig. 2G).
2.4. Preparation and Characterization of PBA-(ZW)-CD/Drug Inclusion Complexes
PBA-(ZW)-CD/drug inclusion complexes were prepared with two chemotherapeutic agents, doxorubicin (DOX) and ulixertinib (UXT) to assess the potential of CRC-targeted combination therapy. Generally, the use of DOX is discouraged in CRC treatment, as DOX activates the extracellular signal-regulated kinase (ERK)-1/2 signaling pathway and induces drug resistance. However, combination therapy with UXT, a potent first-in-class ERK1/2 inhibitor, may help overcome DOX resistance and further enhance therapeutic efficacy (Fig. 3A)59,60,61,62,63. As both drugs were poorly soluble in aqueous media, a co-solvent of dimethyl sulfoxide and water was used to solubilize the drugs with PBA-(ZW)-CD to induce host–guest interactions. After lyophilization, water-soluble PBA-(ZW)-CD/drug inclusion complexes were obtained at molar ratios of drug to PBA-(ZW)-CD of approximately 1 and 2 for DOX and UXT, respectively (Fig. 3B). The drug contents of PBA-(ZW)-CD/DOX and PBA-(ZW)-CD/DOX were 5.10 ± 0.13% and 7.91 ± 0.14%, which were close to the theoretical values of 5.26% and 8.13%, respectively.
The formation of PBA-(ZW)-CD/drug inclusion complexes was investigated using 1H-NMR (Fig. 3C). Although the peaks of CD protons engaging in the host–guest interaction (i.e., protons in the CD cavity) overlapped with strong PEG signals at 3.5–3.9 ppm, we could observe slight changes in the chemical shifts of DOX (red arrows) and UXT (blue arrows) after complexation64,65. These intermolecular correlations were further confirmed by rotating frame Overhauser enhancement spectroscopy (ROESY) (Fig. 3D). The ROESY spectrum of PBA-(ZW)-CD/DOX revealed dipolar couplings between the aromatic proton Ha of DOX and H1 protons of PBA-(ZW)-CD, indicating that the anthraquinone rings of DOX were internalized in the cyclodextrin cavity. The PBA-(ZW)-CD/UXT spectrum also displayed interactions between the protons Hb, Hc, and Hd of UXT and the inner cavity protons H2 and H3 of PBA-(ZW)-CD, supporting inclusion complex formation.
The HDs of the inclusion complexes were measured using DLS to confirm whether PBA-(ZW)-CD maintained its renal-clearable size after drug loading (Fig. 3E). As shown in the number-weighted HD distribution, PBA-(ZW)-CD/DOX had a mean HD of 13.04 ± 3.85 nm at pH 7.4. The slight increase in HD compared to that of the blank PBA-(ZW)-CD could be attributed to concentration-dependent (i.e., reversible) coordination between the primary amine of DOX and boronic acid of PBA-(ZW)-CD66. Since the coordination weakens at acidic pH, the mean HD of PBA-(ZW)-CD/DOX decreased to 7.09 ± 0.26 nm at pH 5.5, which is almost the same value of blank PBA-(ZW)-CD HD (6.58 ± 0.27 nm; Fig. 1C). However, PBA-(ZW)-CD/UXT showed comparable HD distributions at pH 5.5 and 7.4, displaying mean HDs of 6.30 ± 0.11 nm and 6.30 ± 0.09 nm, respectively, which implies the lesser influence of acidity. A similar phenomenon was observed in drug release tests performed at pH 5.5 and 7.4 (Fig. 3F). Due to the DOX–boronic acid coordination, the cumulative DOX release from the inclusion complex reached only 4.9 ± 0.9% even after 24 h incubation at pH 7.4. However, at pH 5.5, the cumulative DOX release significantly increased to 21.3 ± 0.1% (p < 0.005), which could translate into selective drug exposure to tumor tissues, considering the acidic tumor microenvironment (TME). PBA-(ZW)-CD/UXT also showed statistically significant pH-dependency in its release profile, but the cumulative drug release under each condition showed only a 10% difference, displaying 38.9 ± 2.8% and 27.9 ± 1.6% at pH 5.5 and 7.4, respectively. The zeta potential of both inclusion complexes increased at acidic pH due to the ionization of the guest drug while maintaining zwitterionic properties at pH 7.4 (Fig. 3E), which also explains the enhanced drug release of both inclusion complexes at pH 5.5.
2.5. Biodistribution of PBA-(ZW)-CD/Drug Inclusion Complexes
The biodistribution of PBA-(ZW)-CD/drug inclusion complexes was assessed in subcutaneous HT-29 tumor xenograft mice in comparison with that of free drugs, wherein the drug amounts deposited in major organs, muscles, and tumors were determined after intravenous administration of the drug solution or inclusion complex at a dose of 5 mg kg–1. As expected from the biodistribution profiles of PBA-(ZW)-CD/ACy7 (Fig. 1D), DOX exposure to normal tissues (i.e., off-target) in the distribution phase was drastically decreased when treated with PBA-(ZW)-CD/DOX (Fig. 4A). Notably, the DOX amount in the heart at 2 h in the inclusion complex group was 6.5-fold lower than that in the free drug group (p < 0.001). Moreover, in the muscle tissue, the DOX level of the inclusion complex group was below the detection limit (< 200 ng g–1 tissue) even at 0.5 h post injection, while the free DOX group exhibited 2.73 ± 0.30 µg g–1 tissue. Considering that cardiotoxicity and skeletal muscle atrophy are the major side effects of DOX that limit its application to CRC67,68,69,70, the improved biodistribution profile via PBA-(ZW)-CD complexation could enhance the chances of future clinical translation. In the tumor tissue, although both free DOX and PBA-(ZW)-CD/DOX exhibited similar exposure at 0.5 h, PBA-(ZW)-CD/DOX had prolonged tumor retention compared to free DOX, resulting in 2.0-fold higher DOX accumulation at 8 h (p < 0.005).
Unlike DOX, which non-specifically binds to various biomacromolecules, UXT exhibited the expected biodistribution profile when formulated as PBA-(ZW)-CD/UXT (Fig. 4B). PBA-(ZW)-CD/UXT showed rapid clearance in the normal tissues, with UXT amounts remaining in the heart, lungs, kidneys, and liver less than 0.65 µg g–1 tissue within 2 h, which were 8.0-, 17.0-, 5.6-, and 13.6-fold lower values than those of free UXT solution, respectively. In the tumor tissues, however, PBA-(ZW)-CD/UXT displayed a 2.1-fold higher accumulation of UXT than free UXT at 8 h post-injection (p < 0.05). These findings clearly indicate that PBA-(ZW)-CD can enhance the tumor retention of guest drugs and reduce their off-target accumulation in normal tissues, as in the case of PBA-(ZW)-CD/ACy7 (cf. Section 2.2).
Enhancement of the average drug concentration in tumor tissues could be one of the critical elements for successful tumor-targeted drug delivery. However, one cannot underestimate the importance of homogenous drug distribution throughout the tumor mass, as the presence of cancer cells that are poorly exposed to anticancer agents can result in tumor relapse71,72,73. Conventional NCs utilizing the EPR effect suffer from the issue of reduced convection in the TME owing to their large particle size74,75,76,77. PBA-(ZW)-CD/drug inclusion complexes could evade this issue given their ultrasmall size and zwitterionic surface charge. As observed in the ex vivo matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) (Fig. 4C), the intratumoral DOX (m/z 544.6) and UXT (m/z 434.7) signals in the inclusion complex-treated group exhibited a comparable spatial intensity distribution (as the coefficient of variation) to those of the solution-treated group, suggesting that PBA-(ZW)-CD/drug was as highly tumor-penetrable as a free drug solution (Fig. 4D). In addition, the average intensity values of both drugs were significantly higher in the PBA-(ZW)-CD/drug groups than in the corresponding solution groups (Fig. 4E), which correlates well with the biodistribution study performed with tissue homogenates (Fig. 4A and 4B).
The unique pharmacokinetic properties of PBA-(ZW)-CD, which are rarely observed in other clinical reports78, are summarized as follows: (1) PBA-(ZW)-CD may control the biodistribution of cargo molecules in an opposite manner depending on the deposition site, showing decreased and increased clearance in tumor and normal tissues, respectively, and (2) PBA-(ZW)-CD/drug inclusion complexes may penetrate the TME as readily as free drug solutions, promoting homogeneous drug delivery throughout the tumor mass. Based on these findings, we hypothesized that PBA-(ZW)-CD/drug inclusion complexes could simultaneously improve the apparent potency and reduce the potential toxicity of anticancer agents, enabling high-dose administration and subsequent enhancement of therapeutic efficacy.
2.6. PBA-(ZW)-CD/Drug-Assisted Single-Drug and Combination Therapies in HT-29 Xenograft Model
Encouraged by the remarkable biodistribution profiles of PBA-(ZW)-CD, the in vivo antitumor efficacy of each PBA-(ZW)-CD/drug inclusion complex was evaluated in the heterotopic HT-29 xenograft mouse model. As a preliminary study, the in vitro and in vivo toxicity of blank PBA-(ZW)-CD was evaluated. In HT-29 cells, PBA-(ZW)-CD showed negligible cytotoxicity at physiologically-relevant concentrations (~ 500 µg mL–1) (Figure S16A). Moreover, single and repeated (three times) intravenous administration of an excess amount of PBA-(ZW)-CD (270 mg kg–1 per administration) induced no significant deviation in the complete blood counts of mice without observable changes in vital signs or behavioral expressions of pain (Figure S16B).
To evaluate the tumor-suppressive efficiency of PBA-(ZW)-CD/DOX and its potential to reduce the adverse effects of DOX, the dose was set at 5 mg kg–1, wherein significant systemic toxicity and cardiotoxicity of DOX have been reported79,80. As shown in Figure S17, the tumor volume of the PBA-(ZW)-CD/DOX group was significantly smaller than that of the other groups (p < 0.005). The body weight of the DOX solution group was significantly lower than that of the other groups, indicating that a high dose of free DOX caused severe systemic toxicity. However, there was no noticeable change in body weight of the PBA-(ZW)-CD/DOX group during treatment (p < 0.001). Similarly, PBA-(ZW)-CD/UXT also showed a significant improvement in tumor growth inhibition at the therapeutic dose (5 mg kg–1) (p < 0.001), but systemic toxicity in terms of body weight change was not observed in any group (Figure S18). Since the inclusion complex had a marginally higher cytotoxicity than the corresponding free drug (Figure S19), its highly enhanced antitumor efficacy in vivo could mainly result from improved biodistribution.
Next, we performed a combination therapy with PBA-(ZW)-CD/DOX and PBA-(ZW)-CD/UXT in the same mouse model. The mice were divided into six groups, including PBS, blank PBA-(ZW)-CD, DOX or UXT monotherapy (solution), and DOX + UXT combination therapy (solution or inclusion complex), and intravenously injected with each intervention every three days five times, and the doses of DOX and UXT were reduced to 3 mg kg–1, expecting the synergistic effect of DOX and UXT combination. As observed in the tumor growth profile (Fig. 5A), the combination of DOX and UXT solutions exhibited enhanced antitumor efficacy on average when compared to monotherapy with each drug, but no statistically significant difference was found between the DOX + UXT solution and DOX solution groups (p > 0.05). However, the combination of inclusion complexes showed dramatically improved tumor growth inhibition compared to the other groups, of which the average tumor volume on day 14 was 2.1-fold smaller than that in the DOX + UXT solution group (p < 0.05). A similar tendency was observed in the dissected tumor weight data (Fig. 5B), where the PBA-(ZW)-CD/DOX + PBA-(ZW)-CD/UXT group displayed the lowest tumor weight compared to the other groups, showing a 2.9-fold lower average tumor weight on day 14 compared to that of the DOX + UXT solution group (p < 0.05). The superior antitumor efficacy of the PBA-(ZW)-CD/drug combination could be explained by the improved drug distribution to the tumor tissue spatially and temporally enough to induce synergistic effects between DOX and UXT (cf. Section 2.5). Meanwhile, the mice in all groups did not lose body weight significantly during the treatment (Fig. 5C) and showed no abnormal changes in blood biochemistry (Figure S20), indicating negligible systemic toxicity.
2.7. PBA-(ZW)-CD/Drug Combination Therapy in Orthotopic CRC Model
Antitumor efficacy of the PBA-(ZW)-CD/drug-assisted combination therapy was evaluated in an orthotopic CRC model. One week after the inoculation of luciferase-expressing HT-29 (HT-29/Luc) cells into the cecum, the mice were intravenously administered PBS (no intervention), DOX + UXT (solution), or PBA-(ZW)-CD/DOX + PBA-(ZW)-CD/UXT every three days for five times at a dose of 3 mg kg–1 for each drug. Bioluminescence imaging revealed that both solution- and inclusion complex-based combination therapies significantly reduced tumor growth compared to no intervention (Fig. 5D). Notably, PBA-(ZW)-CD/drug combination therapy had a much higher tumor inhibitory efficacy than solution therapy, displaying a 4.2-fold lower bioluminescence intensity on day 14 (p < 0.005) (Fig. 5E).
Unlike the heterotopic model prepared by the subcutaneous injection of HT-29 cell suspension (cf. section 2.6), the orthotopic CRC model prepared via laparotomy exhibited lower tolerability to solution-based combination therapy, with the average body weight reduced to 88.9% of the non-intervention (PBS) group (p < 0.01) (Fig. 5F). However, no weight loss was observed in the PBA-(ZW)-CD/drug combination group, implying that PBA-(ZW)-CD reduced the systemic toxicity of ACT after surgical resection. At the end of the efficacy test, tumor tissues and major organs, including the heart, kidneys, liver, lungs, and spleen, were dissected and subjected to hematoxylin and eosin staining. As shown in Fig. 5G, no detrimental changes were observed in normal tissues, indicating that PBA-(ZW)-CD can minimize the dose-limiting toxicity of anticancer agents. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining was also performed on the tumor tissues (Fig. 5G), which revealed that PBA-(ZW)-CD/drug combination therapy induced a higher degree of apoptosis than solution-based combination therapy. This result correlated well with the antitumor efficacy data (Fig. 5A and 5E).