Development of pH-Responsive Hyaluronic Acid-Conjugated Cyclodextrin Nanoparticles for Chemo-/CO-Gas Dual Therapy

In this study, we fabricated γ-cyclodextrin (γCD)-based nanoparticles (NPs) for dual antitumor therapy. First, γCD (the backbone biopolymer) was chemically conjugated with low-molecular-weight hyaluronic acid (HA; a tumoral CD44 receptor-targeting molecule) and 3-(diethylamino)propylamine (DEAP; a pH-responsive molecule), termed as γCD-(DEAP/HA). The obtained γCD-(DEAP/HA) self-assembled in aqueous solution, producing the γCD-(DEAP/HA) NPs. These NPs efficiently entrapped paclitaxel (PTX; an antitumor drug) and triiron dodecacarbonyl (FeCO; an endogenous cytotoxic gas molecule) via hydrophobic interactions between PTX and FeCO with the unprotonated DEAP molecules in γCD-(DEAP/HA) and a possible host–guest interaction in the γCD rings. The release of PTX and FeCO from the NPs resulted from particle destabilization at endosomal pH, probably owing to the protonation of DEAP in the NPs. In vitro studies using MCF-7 tumor cells demonstrated that these NPs were efficiently internalized by the cells expressing CD44 receptors and enhanced PTX/FeCO-mediated tumor cell apoptosis. Importantly, local light irradiation of FeCO stimulated the generation of cytotoxic CO, resulting in highly improved tumor cell death. We expect that these NPs have potential as dual-modal therapeutic candidates with enhanced antitumor activity in response to acidic pH and local light irradiation.


Introduction
In the development of stimuli-sensitive drug delivery systems, designing site-specific drug carriers by engineering functional biopolymers has often resulted in increasing the local drug dose or maximizing the drug efficacy at specific locations, thus reducing side effects [1][2][3][4]. In particular, the various functionalities of biopolymers or bioactive materials endow drug carriers with attractive physicochemical reactivity and benefit drug carrier reactions with genetic materials, proteins, and biosignals [2,[5][6][7]. Cyclodextrin (CD), a cyclic oligosaccharide, is a known host molecule with a central cavity that allows the inclusion of a guest drug molecule [8][9][10][11]. Remarkably, chemically incorporating functional molecules into CDs is an interesting strategy that strengthens the site-specific bioreaction of CDs, which has often been used to improve drug therapeutic efficacy [10][11][12][13]. Recently, multifunctional CD-based nanoparticles (NPs) have been widely reported for the efficient transportation of antitumor drugs to tumors in response to specific stimuli such as temperature, light, redox, and pH [14][15][16][17][18]. However, such drug carriers have not always shown excellent antitumor effects because they may not overcome the complexities of the cells during the treatment process [16,[19][20][21]. Importantly, it has often been reported that when functional drug carriers transport two or more drugs and respond to multiple stimuli, their multifaceted antitumor effect can sometimes enhance therapeutic outcomes more effectively than a single-drug administration system [16,[19][20][21][22]. outcomes more effectively than a single-drug administration system [16,[19][20][21][22].
In this study, we report dual-modal therapeutic CD-based NPs that respond to both the slightly acidic pH of endosomes and local light irradiation, resulting in the accelerated delivery of paclitaxel (PTX; an antitumor model drug) and carbon monoxide (CO; a cytotoxic model gas) to the local tumor site. PTX can bind microtubules in tumor cells and effectively inhibit cell mitosis, ultimately inducing cell apoptosis [23,24]. CO gas is a potent chemotherapeutic agent that induces tumor cell death through mitochondrial damage. In addition, the hydrophobic prodrug triiron dodecacarbonyl (FeCO) can be degraded under light irradiation, releasing CO gas [25,26]. Therefore, we entrapped both PTX and FeCO in CD-based NPs. We also hypothesized that the CO gas released from FeCO after light irradiation would provide additional antitumor effects, such as CO gasmediated mitochondrial damage in tumor cells, which would effectively enhance PTXmediated cell death. Recent studies have shown that the administration of a combination of multiple drugs with different mechanisms produces a more significant, synergistic antitumor effect and fewer side effects than single-drug therapy [19][20][21][22].
To achieve our goal, we fabricated functional CD-based NPs by conjugating γ-cyclodextrin (γCD) with 3-(diethylamino)propylamine (DEAP; a pH-responsive molecule) [17,18,27] and hyaluronic acid (HA; a CD44 receptor-targeting molecule) [28][29][30][31]. The selfassembly of γCD with DEAP and HA resulted in the formation of γCD-(DEAP/HA) NPs, driven by hydrophobic interactions between DEAP moieties and hydrophilic interactions between γCD and HA. These NPs exhibited organized porosity and enabled multiple interactions between the host (γCD) and guest molecules (PTX and FeCO) [9][10][11]32,33], allowing for the encapsulation of these therapeutic agents. Upon selective internalization into CD44 receptor-expressing tumor cells, the protonated DEAP moieties in the NPs, triggered by the acidic endosomal pH, destabilized the NPs and facilitated the rapid release of PTX and CO gas when exposed to light (Figure 1a). Thus, we focused on investigating the physicochemical properties and release profiles of PTX and CO gas, as well as the in vitro antitumor activity of the γCD-(DEAP/HA) NPs to evaluate their pharmaceutical potential.

PTX and FeCO Loading
γCD-based polymers (100 mg) dissolved in a DMSO (10 mL)/MeOH (10 mL) cosolvent containing PTX (100 mg) and FeCO (80 mg) were stirred for 1 day under dark conditions [17,34]. The resulting solution was dialyzed against DMSO, ultracentrifuged at 20,000 rpm for 30 min, and then lyophilized. Unencapsulated PTX and FeCO were removed by filtration through a 0.2 µm pore size filter [17,18]. The amount of encapsulated PTX in the NPs was confirmed using high-performance liquid chromatography (HPLC, Waters, MA, USA) analysis. For HPLC analysis, the solution was transferred to the mobile phase (acetonitrile/deionized water, 60/40, vol.%), separated using a CAPCELL PAK C 18 column (250 × 4.6 mm, 5 µm, Shiseido Co., Ltd., Tokyo, Japan) at 25 • C, and detected at 227 nm [27]. The PTX or FeCO loading efficiency (%) was calculated as a weight percentage of the PTX or FeCO dose loaded into the NPs to the initial PTX or FeCO fed dose. The PTX or FeCO loading content (%) was calculated as the weight percentage of the encapsulated PTX or FeCO dose to the total amount in the harvested NPs [27,34,35].

In Vitro PTX and CO Release
NPs (with an equivalent PTX concentration of 1 mg/mL or an equivalent FeCO concentration of 100 µg/mL) were dispersed in PBS (150 mM, pH 7.4 or 6.5, 1 mL) and irradiated with a laser at 0 W/cm 2 or 1 W/cm 2 for 10 min. The resulting NPs were added to a dialysis membrane (Spectra/Por ® MWCO 20 kDa) and immersed in PBS (150 mM, pH 7.4 or 6.5, 15 mL) containing 0.01 wt.% sodium azide and 3% Tween 80 [27]. The membranes were incubated in a mechanical shaking bath (100 rpm) at 37 • C for 48 h. A sample of the solution outside of the dialysis bag was removed at specified times and replaced with fresh PBS. The amount of PTX released from the NPs was analyzed using an HPLC instrument [27]. Next, to confirm the CO release profiles from the NPs at pH 7.4~6.5 with and without laser irradiation, the carboxyhemoglobin (HbCO) method was used. Briefly, sodium-dithionite-treated Hb was mixed with light-irradiated NPs in PBS (150 mM, pH 7.4 or 6.5) at 25 • C for 30 min [34,35]. The ultraviolet/visible (UV/Vis) absorption spectra (350-600 nm) of the solutions were monitored at each incubation time using a Cary 1E UV/visible spectrophotometer (Varian Inc., Palo Alto, CA, USA), and the conversion of Hb to HbCO was quantified using Beer-Lambert's law to calculate the CO release from the NPs [34,35].

In Vitro Cellular Uptake
To evaluate the cellular uptake of NPs, NPs were labeled with the fluorescent dye Ce6. Briefly, γCD-based polymers (300 mg) reacted with Ce6 [200 mg, preactivated with ADH (50 mg) for 8 h in DMSO (10 mL) containing DCC (200 mg), NHS (100 mg), and TEA (0.5 mL)] in DMSO (10 mL) containing DCC (100 mg), NHS (50 mg), and TEA (1 mL) for 5 days. To remove unreacted chemicals, the resulting solution was dialyzed using a dialysis membrane (Spectra/Por ® MWCO 1 kDa) for 2 days in DMSO and 2 days in 5 mM sodium tetraborate solution, followed by lyophilization [18]. Here Ce6 conjugation to γCD-based polymers was confirmed by 1 H-NMR analysis [17,18]. We fabricated NPs using fluorescent Ce6 dye-labeled γCD, according to the method mentioned above. The resulting NPs (at an equivalent Ce6 concentration of 10 µg/mL) or free Ce6 (10 µg/mL) were incubated with tumor cells at 37 • C for 4 h. The treated cells were washed with fresh PBS (pH 7.4) three times and analyzed using a FACSCalibur TM flow cytometer (FACSCanto II, Becton Dickinson, Franklin Lakes, NJ, USA). In addition, the cellular distribution of the NPs was visualized in cells stained with DAPI and WGA-Alexa Fluor ® 488 using a confocal laser scanning microscope (LSM710, Carl Zeiss, Oberkochen, Germany) and a hyperspectral camera (CytoViva, Auburn, AL, USA) [17,18].

Statistical Evaluation
All experiment results were analyzed using Student's t-test or ANOVA test with p < 0.01 (**) as a significance level [29,30].
Next, we encapsulated PTX and FeCO (as antitumor model drugs) into γCD-(DEAP/HA) NPs via a dialysis method [17,18]. Here γCD with unprotonated DEAP (pK b~6 .8) selfassembled at pH 7.4 to form a porous γCD core, probably owing to hydrophobic interactions between unprotonated DEAP moieties, while HA segments self-assembled into a hydrophilic shell. Importantly, PTX and FeCO were embedded in γCD-(DEAP/HA) NPs, probably owing to hydrophobic interactions between PTX and FeCO with unprotonated DEAP moieties and possible host-guest interactions in the γCD rings (Figure 1a). However, when the pH of the environment becomes slightly acidic (i.e., endosomal pH), DEAP protonation destabilizes the NPs, weakening the host-guest equilibrium in γCD and thereby accelerating the release of PTX and FeCO [9,10,27,32,33]. In addition, under NIR irradiation, FeCO can be converted into CO gas that attacks the mitochondria of tumor cells (Figure 1a). Based on this hypothesis, we focused on identifying the physicochemical properties of γCD-based NPs and their in vitro antitumor efficacy. Figure S4a shows the particle size and particle morphology of γCD-based NPs at pH 7.4 (normal pH) and pH 6.5 (endosomal pH). At pH 7.4, the NPs had an almost spherical shape, but at pH 6.5, the NPs with pH-responsive DEAP moieties [γCD-(DEAP 7.2 /HA 2.1 ) and γCD-(DEAP 3.4 /HA 2.1 )] became unstable. In addition, under laser irradiation at an intensity of 1 W/cm 2 for 10 min, the morphological changes in the NPs were not significant as shown in Figure S4b. These results indicated that the CO gas generated from FeCO under light irradiation could be easily released from the γCD rings without significantly affecting the structure of the NPs. Figure 2a shows that the particle size of γCD-(DEAP 7.2 /HA 2.1 ) NPs decreased from 146 nm at pH 7.4 to 54 nm at pH 6.5, probably owing to DEAP protonation at pH 6.5 [17,27,30,31]. In γCD-(DEAP 7.2 /HA 2.1 ) NPs, the particle size reduction at pH 6.5 was much greater than that of γCD-(DEAP 3.4 /HA 2.1 ) NPs, revealing the effect of a high DEAP conjugation ratio. In addition, under 1 W/cm 2 laser irradiation for 10 min, the changes in the particle size of all the NPs were not significant. Figure 2b shows that the zeta potential of γCD-(DEAP 7.2 /HA 2.1 ) NPs increased from -28 mV at pH 7.4 to −13 mV at pH 6.5, probably owing to DEAP protonation at pH 6.5 [17,27,30,31]. In addition, the zeta potential changes of γCD-(PA 4.2 /HA 2.0 ) NPs and γCD-(PA 4.2 ) NPs as control groups were not significant, which is similar to their particle size results with no significant difference between pH 6.5 and 7.4 (Figures 2a and S4). Figure 2a shows that the particle size of γCD-(DEAP7.2/HA2.1) NPs decreased from 146 nm at pH 7.4 to 54 nm at pH 6.5, probably owing to DEAP protonation at pH 6.5 [17,27,30,31]. In γCD-(DEAP7.2/HA2.1) NPs, the particle size reduction at pH 6.5 was much greater than that of γCD-(DEAP3.4/HA2.1) NPs, revealing the effect of a high DEAP conjugation ratio. In addition, under 1 W/cm 2 laser irradiation for 10 min, the changes in the particle size of all the NPs were not significant.

In Vitro Release Profiles of γCD-(DEAP/HA) NPs
We investigated the drug release behaviors from the γCD-based NPs at pH 7.4 and 6.5 (Figures 3 and 4) [17,18,27,30,31]. First, the cumulative PTX release from γCD-based NPs at pH 7.4 was approximately 30 wt.% in 24 h, revealing a conventional passive drug release pattern (Figure 3a) [27,30,31]. Under light irradiation (Figure 3b), the γCD-based NPs at pH 7.4 showed similar PTX release as in the case without light irradiation. However, the cumulative PTX release from the γCD-(DEAP 7.2 /HA 2.1 ) NPs and γCD-(DEAP 3.4 /HA 2.1 ) NPs at pH 6.5 averaged 69 and 55 wt.% over 24 h, respectively, probably owing to the DEAP protonation-mediated destabilization of the NPs [17,27,30,31]. In addition, under light irradiation (Figure 3d), the γCD-(DEAP 7.2 /HA 2.1 ) NPs and γCD-(DEAP 3.4 /HA 2.1 ) NPs at pH 7.4 showed similar PTX release as that in the case without light irradiation (Figure 3c).  We also evaluated the amount of CO gas generated from FeCO under light irradiation ( Figure 4). Here the amount of CO gas was calculated using the HbCO method [34,35]. First, the amount of CO gas released from the nonirradiated γCD-based NPs was not significant (Figure 4a). However, the amount of CO gas released from NPs under light irradiation significantly increased regardless of the sample type, suggesting that the generated CO molecules could sufficiently pass through the γCD rings. As a result, our system also signified that NIR could serve as a key stimulus for specific substance release, as demonstrated in a recently published NIR-triggered DOX releasing nanocluster system [36].

In Vitro Cellular Internalization
The cellular uptake of γCD-based NPs by MCF-7 (CD44 receptor-positive) [3 and BT-474 (CD44 receptor-negative) [29] tumor cells was evaluated using flow cytom and confocal laser scanning microscopy. Before testing, each γCD-based NP was lab with the fluorescent dye Ce6; Ce6 was bound at an average molar ratio of 1.1 mole per γCD unit [17,18]. Figure 5a shows that the γCD-based NPs with HA exhibited hi fluorescence intensity in MCF-7 cells than the γCD-based NPs without HA, revealin HA ligand in the γCD-based NPs mediated extensive CD44 receptor interactions. H ever, all NPs showed relatively decreased cellular uptake by BT-474 cells that did no press the CD44 receptor (Figure 5b). In addition, free Ce6, which had no cell-specif teraction abilities, interacted well with both MCF-7 and BT-474 cells. It is known that Ce6 is well absorbed even by normal cells. Moreover, the confocal microscopy im supported that the γCD-based NPs with HA had higher cellular uptake (i.e., a higher signal) in MCF-7 cells than in BT-474 cells, suggesting that γCD-based NPs with HA c effectively target tumor cells with specific CD44 receptors ( Figure 6) [29,37,38]. We evaluated the cellular uptake of PTX and FeCO delivered by γCD-based NPs using perspectral camera [17,18]. The resulting images demonstrated that the γCD-based with HA enabled higher cellular uptake of PTX and FeCO in MCF-7 cells than in BT cells (Figure 7a,b), indicating that the NPs successfully delivered PTX and FeCO MCF-7 cells. We also evaluated the amount of CO gas generated from FeCO under light irradiation (Figure 4). Here the amount of CO gas was calculated using the HbCO method [34,35]. First, the amount of CO gas released from the nonirradiated γCD-based NPs was not significant (Figure 4a). However, the amount of CO gas released from NPs under light irradiation significantly increased regardless of the sample type, suggesting that the generated CO molecules could sufficiently pass through the γCD rings. As a result, our system also signified that NIR could serve as a key stimulus for specific substance release, as demonstrated in a recently published NIR-triggered DOX releasing nanocluster system [36].

In Vitro Cellular Internalization
The cellular uptake of γCD-based NPs by MCF-7 (CD44 receptor-positive) [37,38] and BT-474 (CD44 receptor-negative) [29] tumor cells was evaluated using flow cytometry and confocal laser scanning microscopy. Before testing, each γCD-based NP was labeled with the fluorescent dye Ce6; Ce6 was bound at an average molar ratio of 1.1 molecules per γCD unit [17,18]. Figure 5a shows that the γCD-based NPs with HA exhibited higher fluorescence intensity in MCF-7 cells than the γCD-based NPs without HA, revealing the HA ligand in the γCD-based NPs mediated extensive CD44 receptor interactions. However, all NPs showed relatively decreased cellular uptake by BT-474 cells that did not express the CD44 receptor (Figure 5b). In addition, free Ce6, which had no cell-specific interaction abilities, interacted well with both MCF-7 and BT-474 cells. It is known that free Ce6 is well absorbed even by normal cells. Moreover, the confocal microscopy images supported that the γCD-based NPs with HA had higher cellular uptake (i.e., a higher Ce6 signal) in MCF-7 cells than in BT-474 cells, suggesting that γCD-based NPs with HA could effectively target tumor cells with specific CD44 receptors ( Figure 6) [29,37,38]. We also evaluated the cellular uptake of PTX and FeCO delivered by γCD-based NPs using a hyperspectral camera [17,18]. The resulting images demonstrated that the γCD-based NPs with HA enabled higher cellular uptake of PTX and FeCO in MCF-7 cells than in BT-474 cells (Figure 7a,b), indicating that the NPs successfully delivered PTX and FeCO into MCF-7 cells.

Conclusions
In this study, γCD with a pH-responsive DEAP molecule and tumor CD44 receptortargeting HA was chemically synthesized, and these functional polymers were used to fabricate NPs that effectively delivered PTX and CO gas to tumor sites. These NPs reacted sensitively to endosomal pH and rapidly released PTX and CO gas through the γCD pores or dissociated γCD molecules. This dual-modal therapy exhibited improved antitumor activity compared with the conventional single-drug formulation. This approach based on a functional polysaccharide conjugate is expected to be a novel antitumor drug delivery candidate.