
==== Front
ACS Nano
ACS Nano
nn
ancac3
ACS Nano
1936-0851
1936-086X
American Chemical Society

37717223
10.1021/acsnano.3c04793
Article
Oral Delivery of Gemcitabine-Loaded Glycocholic Acid-Modified Micelles for Cancer Therapy
Zang Wenqing †
Gao Duo ‡
Yu Miaorong ‡
Long Manmei †
Zhang Zhuan ‡
https://orcid.org/0000-0003-3995-455X
Ji Tianhai *†
† Department of Pathology, Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200011, China
‡ State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
* Email: skysea_ji@sina.com.
17 09 2023
26 09 2023
17 09 2024
17 18 1807418088
29 05 2023
14 09 2023
© 2023 The Authors. Published by American Chemical Society
2023
The Authors
https://creativecommons.org/licenses/by-nc-nd/4.0/ Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).

The clinical utility of gemcitabine, an antimetabolite antineoplastic agent applied in various chemotherapy treatments, is limited due to the required intravenous injection. Although chemical structure modifications of gemcitabine result in enhanced oral bioavailability, these modifications compromise complex synthetic routes and cause unexpected side effects. In this study, gemcitabine-loaded glycocholic acid-modified micelles (Gem-PPG) were prepared for enhanced oral chemotherapy. The in vitro transport pathway experiments revealed that intact Gem-PPG were transported across the intestinal epithelial monolayer via an apical sodium-dependent bile acid transporter (ASBT)-mediated pathway. In mice, the pharmacokinetic analyses demonstrated that the oral bioavailability of Gem-PPG approached 81%, compared to less than 20% for unmodified micelles. In addition, the antitumor activity of oral Gem-PPG (30 mg/kg, BIW) was superior to that of free drug injection (60 mg/kg, BIW) in the xenograft model. Moreover, the assessments of hematology, blood chemistry, and histology all indicated the hypotoxicity profile of the drug-loaded micelles.

oral delivery
gemcitabine
micelles
glycocholic acid
chemotherapy
China Association for Science and Technology 10.13039/100010097 2022QNRC001 School of Medicine, Shanghai Jiao Tong University 10.13039/501100008233 JYJC202106 Science and Technology Commission of Shanghai Municipality 10.13039/501100003399 19410740300 China Postdoctoral Science Foundation 10.13039/501100002858 2021M702181 National Natural Science Foundation of China 10.13039/501100001809 91959125 National Natural Science Foundation of China 10.13039/501100001809 81972336 document-id-old-9nn3c04793
document-id-new-14nn3c04793
ccc-price
==== Body
pmcIntroduction

Gemcitabine (Gem), an antimetabolite antineoplastic agent, is widely employed, in combination or alone, to treat a broad range of human cancers (non-small-cell lung carcinoma, metastatic breast cancer, ovarian cancer, and brain cancer, among others),1,2 especially as a first-line therapy in the treatment of pancreatic cancer.3 Because pancreatic cancer exhibits a poor prognosis, due to its high malignancy and low surgical resection rate,4,5 comprehensive treatments, such as chemotherapy, are necessary for pancreatic cancer therapy, especially for late-stage patients. Various chemotherapeutic drugs, such as paclitaxel, 5-FU, gemcitabine, and irinotecan, are widely used for advanced pancreatic cancer.6 Among these drugs, gemcitabine takes priority in terms of improving the overall survival and progression-free survival of cancer patients.7 Gemcitabine is intracellularly converted by deoxy cytidine kinase into its therapeutically active metabolites, the 5′-diphosphate and 5′-triphosphate derivatives, of which the triphosphate derivative can be incorporate into DNA to inhibit its replication and synthesis, ultimately resulting in cytotoxic cell death. However, it is noteworthy that patients commonly receive gemcitabine via the intravenous (i.v.) route, a modality accompanied by notable drawbacks encompassing heightened production expenses and suboptimal patient adherence.8,9 Moreover, this route of administration is prone to eliciting potential adverse effects, including rapid systemic spikes in drug concentration surpassing the threshold of maximum tolerability (MTC), prompting concerns such as abrupt physiological responses and the possibility of air emboli. Furthermore, issues of fluid incompatibility and susceptibility to infections arise due to the substantial fluctuations in plasma drug levels stemming from the rapid elimination kinetics observed following i.v. administration, characterized by a plasma half-life ranging from 8 to 17 min.10 The oral route of drug administration assumes a position of prominence due to its capacity to engender heightened patient adherence and diminished economic burden, resulting in the potential amelioration of toxicological ramifications while concurrently augmenting therapeutic proficiency.11,12 This modality enables the maintenance of sustained and optimal drug concentrations within the systemic circulation, thereby orchestrating an extended period of exposure of malignant cells to the therapeutic agent and thereby precluding the escalation of concentrations beyond the threshold of the MTC. Therefore, the peroral administration of such bioactive pharmaceutical constituents not only proffers a more convenient and comfortable therapeutic avenue for individuals afflicted by cancer but also engenders the capacity to alleviate the strain imposed on healthcare resources within the postpandemic epoch.

Regrettably, gemcitabine manifests an exceedingly limited extent of oral bioavailability (oBA < 10%) stemming from its compromised gastrointestinal (GI) absorption and swift systemic metabolic processes.13 Consequently, the past several decades have witnessed manifold endeavors dedicated to advancing the oral administration paradigm for gemcitabine with a specific emphasis on its sustained release profiles. A noteworthy proportion of these investigative initiatives has been channeled toward the strategic deployment of prodrug formulations, representing a prevailing approach in the pursuit of optimizing gemcitabine’s oral therapeutic viability.14−20 However, the chemical structure modification of gemcitabine commonly attempted, perhaps due to the limiting core structure of gemcitabine (SL-01 with 22.2% oBA and GHC-gemcitabine with 16% oBA in rats), has resulted in the poor enhancement of oral bioavailability.17,20 In addition, some prodrugs may lead to unexpected side effects, especially hepatotoxicity, due to the protection of the metabolic moiety on small molecules.21,22 For example, LY2334737, an orally active N4-valproic acid prodrug of gemcitabine, was developed by Eli Lilly to block the site of deamination to improve the oral bioavailability of gemcitabine hydrochloride in 2009. However, Eli Lilly discontinued the development of LY2334737 in 2013, because it demonstrated hepatic toxicities in patients in the clinical study.23,24 This shows that oral administration of gemcitabine remains an obstacle in clinical applications, so we targeted the development of drug-loaded nanoparticles,25 such as modified micelles, instead of prodrugs to achieve an improved balance between bioavailability and hepatotoxicity. Compared with other carriers, nanoscale micelles offer greater drug stability in the gastrointestinal tract, which provides a platform to permeate through intestinal tract barriers. Besides, micelles can utilize the enhanced permeability and retention effect to support extravasation into the tumor sites.

The efficacious operation of micelles hinges on the judicious selection of polymers employed during the formulation process. The integration of the intestinal bile acid transporter as a strategic avenue has been posited to advance the landscape of oral drug formulations.26−30 A cornerstone of this framework rests upon the enterohepatic circulation of bile acids, a bipartite process encompassing hepatic secretion and intestinal absorption. Within this dynamic milieu, bile acids are secreted into the duodenum to facilitate the absorption of water-insoluble nutrients via emulsification.31 Notably, bile acids undergo absorptive processes encompassing both passive and active mechanisms within the distal segments of the small intestine. Specifically, passive absorption predominates within the proximal extents of the small intestine and colon, while active absorption is confined to the anatomical precincts of the ileum.32 In higher organisms, including humans, intricate and efficient transport mechanisms have evolved within the terrain of the ileal epithelium, enabling the active reclamation of bile acids, thus culminating in an efficient enterohepatic circulation characterized by the excretion of a mere 5–10% of intestinal bile acids through the fecal route.33,34 Empirical inquiries have substantiated the nanoparticles, intricately conjugated with bile acid derivatives, engage in interactive interfaces with the bile acid transporters ensconced within the membranes of the small intestine,35,36 which engenders a conducive milieu for facilitating the uptake of drugs within the enterohepatic milieu.

Briefly, the enterohepatic circulation of bile acid has attracted great attention, and nanoparticles modified with glycocholic acid (GCA) have exhibited promise in becoming oral drug delivery carriers in recent years, with their enhanced oral bioavailability and hypotoxicity.37 GCA, an integral derivative of bile acid, assumes a preeminent role as the principal constituent within the spectrum of human bile salts.38 Distinguished by its notably diminished logarithmic partition coefficient (logP), GCA demonstrates an inherent propensity for greater interaction with the aqueous phase, owing to its reduced lipophilicity.39 Therefore, GCA is more effectively absorbed and transported by the bile acid transporter (apical sodium-dependent bile acid transporter, ASBT) on the small intestine membrane. Moreover, the surface modification of micelles using GCA can greatly promote the active transport and absorption of drug-loaded micelles in the small intestine. Bae et al. examined Ex-4-loaded glycocholic-acid-coated liposomes to control type 2 diabetes in 2019,40 and the oral bioavailability of small molecular drugs received a substantial improvement. Although the oral administration of Ex-4 was realized, the relatively unfavorable oral bioavailability (oBA = 19.5%) limited the application of such liposomes in oral chemotherapeutic drugs due to the requirement of higher plasma concentration and area under the curve (AUC) to have an antitumor effect.

Against this contextual backdrop, we present empirical substantiation demonstrating the amplified potential for oral gemcitabine delivery by means of drug-incorporated micelles fashioned from GCA-modified poly(lactic-co-glycolic acid)–poly(ethylene glycol) (PLGA–PEG) copolymers, denoted as PPG, as opposed to the conventional employment of liposomes. The synthetic method for the target polymer PLGA–PEG–GCA required only two steps of amide condensation. We compared the pharmacokinetic and pharmacodynamic efficacy of Gem-PPG with unmodified micelles and i.v.-injected free drug solution. Not only good oral bioavailability but also superior anti-pancreatic cancer activity and hypo-hepatotoxicity profiles of Gem-PPG micelles were achieved simultaneously, which overcame the limitations (e.g., low oBAs or unacceptable hepatic toxicities) of traditional gemcitabine prodrugs.

Results and Discussion

Synthesis and Characterization of PPG Polymer Material

In pursuit of enhancing the bioavailability of the hydrophobic therapeutic agent gemcitabine and facilitating its intestinal absorption, we engineered a glycocholic acid-conjugated PLGA–PEG polymer, designated as PPG. The PLGA–PEG polymer is FDA approved to prepare nanodrugs because of its favorable biocompatibility and stability.41 A meticulously devised synthetic protocol for PPG is graphically represented in Figure 1a. This synthetic route involved the transformation of the carboxyl group of GCA into a primary amine via a reaction with ethylenediamine (EDA), resulting in the production of GCA–EDA. Subsequently, the modified GCA was conjugated to the carboxylic groups of PLGA10k–PEG5k–COOH (PP) through a conventional carbodiimide coupling reaction. Confirmation of successful PPG synthesis was substantiated by the appearance of distinctive −CH3 peaks corresponding to the GCA at 0.58 ppm, as evidenced by 1H NMR spectroscopy. The extent of GCA conjugation onto PLGA–PEG was quantified by comparing the intensity of the −CH3 peak of PLGA–PEG at a chemical shift of 1.47 ppm to that of GCA at 0.58 ppm. This analysis revealed a degree of GCA conjugation equating to approximately 67 GCA units per 100 monomeric units, signifying that a substantial majority of the carboxylic groups had been successfully modified by GCA (Figure 2e). Supplementary 1H NMR spectra for GCA and PLGA10k–PEG5k–COOH are available in Figure S1.

Figure 1 (a) Conjugation of GCA to carboxylate PEG–PLGA. (b) Schematic representation of gemcitabine-loaded PLGA–PEG–GCA (Gem-PPG) micelles.

Figure 2 Size and morphologies of gemcitabine-loaded micelles. The TEM image (a) and size distribution (b) of Gem-PPG60. The TEM image of Gem-PPG60 shows formation of spherical nanosized particles. The TEM image of Gem-PPG100 (c) and Gem-PP100 (d). Scale bar, 100 nm. (e) 1H NMR spectra of PLGA–PEG–GCA polymer. (f) The in vitro release of 60 and 100 nm gemcitabine-loaded micelles (mean ± SD, n = 3).

Preparation and Characterization of Gemcitabine Micelles

Four micelles shown in Table 1 were prepared by ultrasound-assisted emulsification using the synthetic polymers. This method has been shown to yield a uniform size and narrowed distribution.42 The schematic representation of Gem-PPG micelles is presented in Figure 1b. The size distribution, zeta potential, and surface density of different gemcitabine micelles are summarized in Table 1. The zeta potential of the Gem-PPG60 and Gem-PPG100 changed from −45.2 and −48.7 mV to −23.2 and −21.3 mV, respectively, attributed to the anionic carboxyl of PP being modified by GCA. Corresponding to the degree of GCA conjugation, the GCA surface densities of Gem-PPG60 and Gem-PPG100 micelles were 161/230 and 608/862 per micelle, respectively (Figures S2, S3). The gemcitabine loading capacities of Gem-PPG60, Gem-PPG100, and unmodified micelles were 11.7, 11.2, and 9.6 wt %, respectively, while the entrapment efficiencies of gemcitabine in these micelles were 71.2, 72.1, and 64.9, respectively, which demonstrated the better drug loading character of GCA-modified micelles (Table 2). The size and shape of Gem-PPG60, Gem-PPG100, and Gem-PP100 were measured by transmission electron microscopy (TEM) after negative staining by 1% uranium acetate. All of the micelles exhibited relatively uniform spherical structures, which confirmed the efficiency of the ultrasonic emulsification method for preparing the micelles (Figure 2a–d and Figure S4).

Table 1 Particle Size, Zeta Potential, and Surface Density of COOH and GCA of the Gemcitabine-Loaded Micelles (n = 3, Mean ± SD)

formulation	size (nm)	PDI	zeta-potential (mV)	of COOH per micelle	of GCA per micelle	
Gem-PPG60	66.32 ± 4.16	0.062	–23.2 ± 0.7	69	161	
Gem-PP60	63.13 ± 5.74	0.180	–45.2 ± 3.6	230	0	
Gem-PPG100	120.79 ± 9.17	0.086	–21.3 ± 0.5	254	608	
Gem-PP100	114.42 ± 6.31	0.110	–48.7 ± 2.1	862	0	

Table 2 Entrapment Efficiency and Loading Capacity of the Gemcitabine-Loaded Micelles

formulation	entrapment efficiency (%)	loading capacity (%)	
Gem-PPG60	71.2	11.7	
Gem-PPG100	72.1	11.2	
Gem-PP100	64.9	9.6	

In Vitro Gemcitabine Micelle Release Pattern

The micelle release profiles were then conducted in vitro. Notably, gemcitabine-loaded micelles devoid of glycocholic acid modification demonstrated a notably rapid drug release profile. Specifically, within the initial 24 h, 47.1 ± 3.0% of the encapsulated gemcitabine permeated the dialysis membrane, a proportion that extended to 62.5 ± 3.2% after 120 h. In contrast, gemcitabine-loaded PPG micelles exhibited a notably sustained release spanning the entire 120 h observation period, with Gem-PPG60 releasing 42.7 ± 2.1% of the drug and Gem-PPG100 releasing 57.2 ± 3.1% (Figure 2f). Of noteworthy significance is the realization that Gem-PPG60 attains the most protracted and durable release pattern, an attribute with consequential clinical implications. This characteristic is particularly advantageous, as a substantial portion of the drug remains sequestered within the micellar formulation and circulates within the physiological milieu (pH 7.4), thus potentially augmenting drug accumulation at tumor sites via the enhanced permeability and retention (EPR) effect intrinsic to nanoparticle-based drug delivery systems.43

Stability and Release Pattern of Gemcitabine-Loaded Micelles in Simulated Gastrointestinal Fluids (GIF)

The particle properties were evaluated in simulated GIF. The size and surface zeta potentials of different micelles were stable under simulated GIF (Figure 3a,b,d,e). The variation of zeta potentials of Gem-PPG60 and Gem-PPG100 in the GIF was smaller than that of Gem-PP100, indicating that GCA modification facilitates the stability of micelles. The gemcitabine release profiles were also explored in GIF. Gem-PPG60, Gem-PPG100, and Gem-PP100 micelles exhibited 4.0%, 7.9%, and 7.8% cumulative release in simulated gastric fluid (pH 1.2) at 3 h (Figure 3c). In simulated intestinal medium (pH 6.8) at 6 h, the release efficiencies of Gem-PPG60, Gem-PPG100, and Gem-PP100 were only 6.4%, 12.7%, and 12.5%, respectively (Figure 3f). These findings collectively underscore the robust stability of all micellar formulations within the gastrointestinal context and their capacity to release only modest quantities of gemcitabine during their passage through the gastrointestinal tract. Importantly, these release characteristics hold promise in enhancing drug absorption, as they extend the window of opportunity for gemcitabine-loaded micelles to reach the distal regions of the small intestine, potentially enhancing therapeutic efficacy.

Figure 3 Size, surface zeta potentials, and cumulative drug release profiles of Gem-PPG60, Gem-PPG100, and Gem-PP100 micelles in the simulated gastric fluid (pH 1.2, a–c) and simulated intestinal fluid (pH 6.8, d–f).

In Vitro Cellular Uptake through ASBT-Mediated Endocytosis of PPG Micelles

Caco-2 cell monolayers, capable of expressing the ASBT, served as an in vitro model to replicate the features of the intestinal epithelium. The qualitative assessment of the internalization of 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO)-loaded micelles by these Caco-2 cell monolayers was conducted through the application of confocal laser scanning microscopy (CLSM). In this context, the PPG60 group exhibited significantly enhanced green fluorescence intensity, in contrast to both the phosphate-buffered saline (PBS) solution (used as a control) and the DiO-PP100 groups, as visually depicted in Figure 4a. To quantitatively evaluate the extent of cellular uptake by Caco-2 cells, a comprehensive analysis was carried out. As depicted in Figure 4b, the quantity of DiO fluorochrome internalized into cells incubated with DiO-PPG60 micelles significantly exceeded that observed in cells treated with DiO-PP100 micelles. These findings underscore the heightened cellular uptake capacity of DiO-PPG60 micelles relative to those of their DiO-PP100 counterparts.

Figure 4 Endocytosis of micelles via the ASBT-mediated apical pathway. (a) CLSM images illustrating Caco-2 cell monolayers following a 1 h incubation with DiO-labeled PP100 micelles and PPG60 micelles. The cell nuclei were counterstained with DAPI (blue). (b) Quantitative analysis depicting the cellular uptake quantities associated with different treatments. (c) Visualization of ASBT (appearing in red) distribution within cellular structures subsequent to exposure to DiO-loaded PP100 micelles and PPG60 micelles. (d) Examination of the apparent permeability coefficient (Papp) for micelles traversing Caco-2 cell monolayers. The control group represents Caco-2 cells incubated with PBS. Cell nuclei were stained with DAPI (blue). Data are presented as mean ± SD (n = 3), ***p < 0.001, ****p < 0.0001. Scale bar, 20 μm. (e) The TEM images of PPG60 micelles after transport. Scale bar, 200 nm.

To validate the involvement of ASBT in mediating the intracellular uptake of PPG60, we conducted colocalization investigations, examining the spatial relationship between ASBT and micelles within Caco-2 cell monolayers by employing CLSM. As visually demonstrated in Figure 4c, ASBT was localized to the cytomembrane of Caco-2 cell monolayers. Notably, upon treatment with DiO-PP100, minimal colocalization events were observed. In stark contrast, robust yellow fluorescence signals emanated from the cytoplasm after DiO-PPG60 treatment, indicative of a heightened interaction between GCA-modified micelles and ASBT receptors. These observations compellingly support the notion that cellular internalization of PPG60 micelles was achieved through the ASBT-mediated endocytic pathway.

In Vitro Nanoparticle Transport Efficacy Using the Caco-2 Model

We conducted an evaluation of the transepithelial permeability of micelles across the Caco-2 cell monolayers. The results, as depicted in Figure 4d, reveal that the apparent permeability coefficient (Papp) for DiO-PPG60 micelles registered at 11.44 × 10–6 cm/s, demonstrating a noteworthy 2.2-fold increase in comparison to the Papp value observed for unmodified DiO-PP100 micelles, which stood at 5.27 × 10–6 cm/s. This indicates the enhanced transepithelial permeability of GCA-modified micelles. In addition, the TEM image of PPG60 micelles exhibited the similar and intact structure of nanoparticles after transport of the Caco-2 cell monolayers (Figure 4e).

In Vivo Pharmacokinetic Study

Pharmacokinetic investigations were systematically undertaken to ascertain the oral bioavailability of Gem-PPG60, Gem-PPG100, and their unmodified counterpart, Gem-PP100. Detailed pharmacokinetic parameters are comprehensively outlined in Table 3. To assess the plasma levels of gemcitabine, a series of experiments involving oral and intravenous administration of various gemcitabine-loaded micelle formulations were conducted in rats, as depicted in Figure 5a. Upon oral administration, it was unequivocally evident that both GCA-modified micelle formulations yielded an augmentation in bioavailability, approximately 3- to 4-fold higher when juxtaposed with their unmodified counterparts. In particular, Gem-PPG60 exhibited a noteworthy maximum drug concentration (Cmax) of 22 ± 2.3 μg/mL, a pinnacle achieved at the 4 h mark subsequent to oral administration. Notably, this Cmax value significantly exceeded that observed with Gem-PP100, which recorded a Cmax of 5 ± 0.5 μg/mL. Additionally, the AUC value of Gem-PPG60 was 134 ± 12 μg·h/mL, compared to only 32 ± 3 μg·h/mL for Gem-PP100, which indicates the increased absorption through GCA modification. Moreover, the half-lives of orally administered Gem-PPG60 and Gem-PPG100 were 6.13 ± 0.1 and 6.97 ± 0.3 h, respectively, compared to that of i.v. administration (0.53 ± 0.06 h), which suggests the superior metabolism portfolio of oral administration. Furthermore, the absolute oral bioavailability of Gem-PPG60 (80.7%) was 1.2 times and 4.2 times higher than that of Gem-PPG100 (68.7%) and Gem-PP100 (19.3%), respectively. But also, the micelles’ oral bioavailability (80.7%) was better than that of previously reported prodrugs, such as SL-01 (22.2%) and GHC-gemcitabine (16%), in rats.15,18

Table 3 Pharmacokinetic Analysis of i.v. Injection of Gem-PPG100 Micelles and Oral Administration of 60 and 100 nm Gem-PPG Micelles and Unmodified Gem-PP100 Micelles in Normal Rats (Mean ± SD, n = 3)

PK parameters	Gem-PPG100 micelles (i.v.)	Gem-PPG100 micelles (oral)	Gem-PPG60 micelles (oral)	Gem-PP100 micelles (oral)	
Cmax (μg/mL)	142 ± 6.7	21 ± 0.0	22 ± 2.3	5 ± 0.5	
Tmax (h)	0.0	4.0 ± 0.0	4.0 ± 0.0	4.0 ± 0.0	
AUC0-inf (μg·h/mL)	166 ± 21	114 ± 21	134 ± 12	32 ± 3	
T1/2 (h)	0.53 ± 0.06	6.97 ± 0.30	6.13 ± 0.10	6.33 ± 0.30	
bioavailability (F%)	100	68.7	80.7	19.3	

Figure 5 (a) Plasma gemcitabine level versus time profiles of SD rats. Gem-PPG100 (10 mg/kg) was administered by i.v. injection and oral gavage, respectively. Gem-PPG60 and Gem-PP100 (10 mg/kg) were administered by oral gavage. (b) Biodistribution of isolated main organs 8 h postadministration of 10 mg/kg gemcitabine solution and 10 mg/kg Gem-PPG60 by i.v. and orally into SD rats, respectively (mean ± SD, n = 3). *p < 0.05, **p < 0.01.

The GCA-modified micellar formulations have demonstrated a significant improvement in oral bioavailability compared with their unmodified counterparts. These findings are aligned with the outcomes of invitro permeability investigations, wherein the micellar delivery system effectively enhances the transintestinal transport of the drug candidate across the epithelial membrane, in contrast to unmodified micelles. The absorption enhancement of GCA-modified micelles was more obvious in vivo than that in the Caco-2 model, due to the main ASBT distribution on the ileum.44 Moreover, following administration of the drug solution, the drug that reached systematic circulation was rapidly metabolized, resulting in a short plasma half-life.45 Conversely, the drug-loaded micelles served as an effective safeguard, effectively shielding the drug from in vivo metabolism. This protective effect resulted in the sustained release of gemcitabine, thereby significantly prolonging the drug’s half-life to approximately 6.13 h. Taken together, these results explain the in vivo data observed in Figure 5a, for which the 60 nm GCA-modified drug-loaded micelles take priority over others.

Biodistribution Study

We then performed a biodistribution study to explore whether the micelles could enhance the accumulation of gemcitabine in the targeted tissue. The biodistribution levels of gemcitabine micelles in the heart, liver, spleen, and lung were relatively higher than that of the drug solution at 8 h, which indicates the superior drug aggregation of the oral micelles in vital organs (p < 0.05, respectively). In addition, the level of i.v. drug solution in the pancreas was lower (p < 0.05) than that of oral micelles; the mean value of gemcitabine in the pancreas exhibited a highest 2.56-fold change, which suggests a superior level of Gem-PPG60 distribution in the pancreas to take effect in vivo (Figure 5b). As a result, the pancreatic cancer model was considered in the following pharmacodynamics studies.

In Vitro Efficiency of Gemcitabine-Loaded Micelles against Pancreatic Cancer Cells

As pancreatic cancer has been preferred for gemcitabine treatment, the antitumor efficacy of the gemcitabine-loaded micelles alongside the gemcitabine solution was determined in both BxPC-3 and Mia-Paca-2 pancreatic cancer cells. Gem-PPG60, unmodified Gem-PP100, and gemcitabine solution demonstrated dose-dependent toxicity over 120 h, with GI50s of 5.3, 5.8, and 4.0 nM, respectively, in BxPC-3 and 11.0, 11.9, and 7.3 nM, respectively, in Mia-Paca-2 (Figure 6a,b). The GI50s of GCA-conjugated micelles were relatively lower than those of unconjugated micelles, which indicates the effectiveness of glycocholic acid modification on micelles. Additionally, it is worth noting that both drug-loaded formulations demonstrated slightly elevated GI50 values compared to a gemcitabine solution. This phenomenon can be attributed to the encapsulation of the drug within the carrier systems. The GI50s of micelles without drug loading were evaluated by the same method (7.3 μM in BxPC-3 cells and 16.5 μM in Mia-Paca-2 cells), which indicates the favorable biocompatibility of the PPG polymer (Figure S5). In addition, Gem-PPG60 exhibited potential antitumor activity in U87 cells, with a GI50 of 5.1 nM (Figure S6), which suggests its potential in glioma treatment, due to the similar cell toxicity of pancreatic cancer. Nevertheless, further exploration was restricted by the low blood–brain barrier permeability in vitro (Table S2).

Figure 6 In vitro efficiency study of gemcitabine solution and 60 and 100 nm gemcitabine micelles on BxPC-3 (a) and Mia-Paca-2 (b) pancreatic cancer cells encompassing a range of concentrations over a 5-day experimental duration (mean ± SD, n = 3). (c) In vivo investigations entailed the acquisition of representative fluorescence images at varying time intervals, postadministration (dose: 20 μg DiR/kg). (d) Ex vivo ICG fluorescence images and (e) quantitative determination of the organs and tumors excised from the micelle group after 48 h.

In Vivo Pharmacodynamics with BxPC-3 Tumor Mice

Due to the relative advantage offered with respect to cellular toxicity, drug release, and oral bioavailability, 60 nm micelles were selected. First, we examined the in vivo imaging of fluorescent dye-DiR-loaded 60 nm PPG micelles (DiR-PPG60), using an animal model bearing BxPC-3 cancer cells. Bioluminescence imaging was followed by the development of a xenograft model. Four hours following the oral administration of DiR-PPG60, discernible optical imaging signals registered an augmentation specifically within the tumor site, situated atop the right hind leg. By the 24 h postadministration mark, the most robust fluorescence intensities attributable to DiR-PPG60 micelles were unequivocally manifested within the tumor milieu, demonstrating that PPG60 micelles reached a high level of BxPC-3 tumor aggregation and peaked in the tumor. Moreover, this heightened optical imaging signal within the tumor region persisted even 48 h after oral administration of the micelles (Figure 6c). The fluorescence signal and quantitative determination from excised organs and tumors confirmed that the drug concentrated in the BxPC-3 tumor and some metabolic organs (liver and spleen) (Figure 6d,e).

The efficacy of micelles in pancreatic cancer therapy was further explored in vivo with a BxPC-3 xenograft model. When the tumor volume reached 98–136 mm3, saline (oral, BIW, negative control), gemcitabine hydrochloride injection (i.p., 60 mg/kg, BIW, positive control), and Gem-PPG60 micelles (oral, 30 mg/kg, BIW) were administered to the tumor-bearing mice. Consequent changes in tumor volume were meticulously monitored and documented. The results, as visually presented in Figure 7a, elucidate discernible disparities in tumor progression among the experimental cohorts. In particular, the tumor volume in the saline control group exhibited a notable increase over time. In stark contrast, the Gem-PPG60 micelle treatment group displayed a markedly reduced tumor growth rate (p < 0.05), resulting in a 2.3-fold reduction in tumor volume when compared to the control group that received no treatment. This effect persisted until the 33rd day of the study, thereby underscoring the substantial therapeutic efficacy of Gem-PPG60 micelles in inhibiting the progression of BxPC-3 pancreatic tumors. As shown in Figure 7b, no significant body weight variation of the mice in all three groups was observed, which illustrates the biocompatibility of the Gem-PPG60 micelles. Figure 7c depicts the solid tumors, with the saline control group and the 60 mg/kg drug solution and 30 mg/kg micelle groups all harvested on day 33. Moreover, the 30 mg/kg gemcitabine micelle group (TGI = 68.1%) exhibited a better antitumor effect than the 60 mg/kg gemcitabine hydrochloride solution group (TGI = 49.1%) after a 33-day treatment, which demonstrates the more effective antitumor portfolio of orally administered Gem-PPG60 micelles compared to commercially available gemcitabine hydrochloride injection. Ki-67 immunostaining confirmed the superior inhibition of cancer cell proliferation by micelles, with a Ki-67 ratio for the micelle group (0.39) lower than that for the drug injection group (0.63) (Figure 7d and Figure S7).

Figure 7 (a) The temporal evolution of tumor size in BALB/c mice was meticulously documented throughout the 33-day treatment period involving gemcitabine solution (i.p., 60 mg/kg, BIW), Gem-PPG60 micelles (oral, 30 mg/kg, BIW), and the untreated group (saline group, oral, BIW). (b) The pattern of body weight alteration in BALB/c nude mice was comprehensively monitored over the course of the 33-day treatment regimen. (c) Photographic documentation of the harvested solid tumors, obtained from the respective experimental groups: saline control, 60 mg/kg drug solution, and 30 mg/kg drug-loaded GCA micelles. These interventions were administered twice weekly, and tumor harvesting occurred on the 33rd day (mean ± SEM, n = 5, #p < 0.1 *p < 0.05, **p < 0.01). (d) Immunohistochemical evaluation of tumor tissues extracted from mice subjected to various treatments, including saline control, gemcitabine solution, and Gem-PPG60 micelles. The immunostaining focused on the proliferation marker Ki-67. Scale bar, 100 μm. Representative microphotographs and quantitative analysis of Ki-67 immunostaining against tumor tissues are presented (n = 3, ***p < 0.001).

Hematology, Blood Chemistry, and Histology Studies

The hematology and blood chemistry indexes were evaluated on the 33rd day. Although the WBC and RBC levels of the Gem-PPG60 micelle group decreased to 6.23 × 109/L and 7.57 × 1012/L, respectively, which were lower than those of the saline group (8.30 × 109/L and 8.72 × 1012/L), no statistically significant variation was observed between the micelle and commercial gemcitabine injection groups. Moreover, the PLT concentration of the micelle group increased to 816.7 × 109/L (Figure 8a–c). This indicated that orally delivered micelles caused no more serious hematotoxicity than drug injection. However, the mean values of ALT (24.0 U/L) and AST (209.7 U/L) levels in the gemcitabine injection group were higher than others (1.48- and 1.84-fold, respectively), which suggests hepatic injury of the free gemcitabine formulation (Figure 8d,e). In contrast, the CRE level of gemcitabine micelles increased to 18.7 μmol/L, which indicated the probability of renal injury by micelles (Figure 8f).

Figure 8 Comparison of (a) WBC, (b) RBC, (c) PLT, (d) ALT, (e) AST, and (f) CRE levels after 33 days of treatment with saline, gemcitabine solution, and Gem-PPG60 on BALB/c nude mice (mean ± SD, n = 3).

Histological investigations were carried out to assess the extent of organ damage within each experimental group. Representative photomicrographs depicting the histology of multiple organs, including the heart, liver, spleen, lung, kidney, pancreas, esophagus, stomach, ileum, and colon, are visually presented in Figure 9. These visual representations were captured following a 33-day treatment regimen encompassing the administration of saline, a commercial gemcitabine hydrochloride injection, and oral Gem-PPG60, respectively. No major evidence of toxicity was discernible in the saline and Gem-PPG60 micelle groups. However, in the livers of the drug injection group, a low degree of inflammation was observed. Moreover, in the portal area (black arrows) and around the central vein, piecemeal hepatocyte degeneration and lysis with bleeding were observed (red arrows), and the liver plate structure was disordered, without normally radial arrangement (blue arrows), which indicates direct hepatic toxicity by gemcitabine (Figure 10). These findings imply that the hepatotoxicity associated with the unformulated gemcitabine injection was notably more severe when compared to that of gemcitabine encapsulated within the PPG micelles. Additionally, it is noteworthy that there was an absence of observable macroscopic nanoparticle remnants within the examined organs, thereby affirming the consistent biodegradability of the PLGA–PEG–GCA polymer. Moreover, no major signs of toxicity were observed in other organs, especially the kidney, illustrating the transient injury of the kidney instead of continuous damage and would not lead to organic pathological changes of the kidney, despite the raised creatinine level in the Gem-PPG60 group. Furthermore, the biocompatible profile in GIT organs, such as the esophagus, stomach, ileum, and colon, demonstrates the feasibility of the drug-loaded micelles through oral delivery.

Figure 9 Representative photomicrography (×20) of the heart, liver, spleen, lung, kidney, pancreas, esophagus, stomach, ileum, and colon with H&E staining following dosing with saline, gemcitabine solution, and gemcitabine-loaded PPG micelles on BALB/c mice. Scale bar, 100 μm.

Figure 10 Representative photomicrography (×20) of the liver with H&E staining following dosing with saline, gemcitabine solution, and gemcitabine-loaded PPG micelles on BALB/c mice (n = 3). Scale bar, 100 μm.

Conclusions and Prospects

To assess the clinical applicability of gemcitabine oral delivery via bile acid transporters, we engineered micelles incorporating glycocholic acid modifications for the encapsulation of gemcitabine. The GCA moiety facilitates the oral absorption of the micelles through ASBT receptors on the ileum, while the PLGA–PEG moiety provided biocompatibility and a sustained release profile. The excellent oral bioavailability, superior antitumor efficacy, and good hepatotoxicity profiles of these micelles support their preliminary safety and effectiveness for treating pancreatic cancer in mice. The 60 nm micelles exhibited higher oral bioavailability than the 100 nm ones perhaps due to their superior endocytosis efficiency.

Further evaluations and drug safety assessments, such as reproductive toxicity, genotoxicity, and carcinogenicity, will be conducted in addition to evaluating the acute and chronic toxicity of the micelles. The APIs loaded in the micelle could be switched with other classically hydrophobic drugs, such as paclitaxel, cisplatin, and PROTACs due to the hydrophobic effect between these hydrophobic compounds and the PLGA moiety of the micelles, to realize their oral administration and expand potential indications. Moreover, encouraged by the excellent antitumor efficiency on glioma cells of the drug in vitro, a method named amphipathic-multiple modification has been established to further transform the micelles to overcome the blood–brain barrier and establish an oral brain cancer therapy. Furthermore, more evidence of the safety and efficiency patterns for cancer patients of the micelles will be acquired through clinical studies after preclinical preparation.

Materials and Methods

Materials

GCA, dicyclohexylcarbodiimide (DCC), ethylenediamine (EDA), N-hydroxysuccinimide (NHS), ethyl acetate, dimethylformamide (DMF), and methanol were purchased from Aladdin (Shanghai, China). 1-(3-(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC), dimethyl sulfoxide (DMSO), and DMSO-d6 were purchased from Bidepharm (Shanghai, China). PLGA10k and PLGA10k–PEG5k–COOH were purchased from Tanch-Tech (Guangzhou, China). Gemcitabine was purchased from Selleck Chemicals (Shanghai, China), and a gemcitabine hydrochloride solution was obtained from CTTQ Pharma (Nanjing, China). ASBT goat polyclonal antibody was obtained from Santa Cruz Biotechnology, Inc. (Texas, USA). 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and Cy3-labeled donkey anti-goat IgG were purchased from Beyotime Biotechnology (Shanghai, China). Transwell inserts were bought from Corning (New York, USA).

Two-Step Synthesis of Glycocholic Acid-Conjugated PLGA–PEG Polymer

The synthesis of PPG was conducted following previously reported procedures.44 For the synthesis of GCA-EDA, GCA (500 mg, 1.0 equiv), DCC (160 mg, 1.3 equiv), and EDA (3.2 g, 50 equiv) were dissolved in 10 mL of dry DMF. The resulting reaction mixture was stirred for 24 h at 35 °C. Subsequently, the mixture underwent filtration, and any residual EDA was eliminated under vacuum conditions. The concentrated solution was then precipitated using ethyl acetate, and the obtained precipitate was subjected to washing with ethyl acetate and subsequent drying under a vacuum for 24 h, yielding GCA–EDA in powdered form.

To synthesize PPG, PLGA10k–PEG5k–COOH (600 mg, 1.0 equiv), EDC (15.3 mg, 2.0 equiv), and NHS (9.2 mg, 2.0 equiv) were combined in 10 mL of dry DMSO under N2 protection. The mixture was agitated for 4 h at 30 °C to activate the carboxyl moiety. Subsequently, GCA–EDA (60.8 mg, 3.0 equiv) and 10 μL of distilled EDA were introduced, and the reaction mixture was stirred for an additional 10 h. The resulting crude product underwent purification through dialysis in methanol (with a molecular weight cutoff of 1000 Da), followed by drying under vacuum. The purified product was then redissolved in 10 mL of distilled water and subjected to lyophilization to obtain the final product. The synthesized PPG was characterized by using a Varian Unity 400 1H NMR spectrophotometer.

Preparation of Gemcitabine-Loaded PLGA–PEG–GCA Micelles (Gem-PPG)

Gem-PPG micelles were prepared by ultrasound-assisted emulsification.46

For 100 nm Gem-PPG micelles (Gem-PPG100), gemcitabine (3.0 mg) and synthesized PPG (30.0 mg) were dissolved in 1 mL of DMSO. The mixture was added to 20 mL of distilled water dropwise under ultrasound in 5 min to produce an emulsified mixture. The purification of PPG micelles ensued through a meticulous dialysis procedure employing a membrane with a molecular weight cutoff of 14 kDa, conducted using distilled water for an extended duration of 24 h, with the primary aim of eliminating any residual, unencapsulated gemcitabine. Subsequently, the raw micellar solution underwent a filtration process facilitated by a 450 nm filter (Millipore, USA), followed by ultrafiltration at a rapid rotational speed of 1500 r/min, effectively eliminating any remnants of the PPG polymer component. Last, the mixture was passed through a 220 nm filter (Millipore, USA) to remove aggregation, and the purified Gem-PPG100 micelles were stored at 4 °C. For DiO-labeled micelles, the same method was employed, with gemcitabine being replaced by DiO.

For 60 nm Gem-PPG micelles (Gem-PPG60), gemcitabine (6.0 mg), PLGA10k (6.0 mg), and synthesized PPG (30.0 mg) were dissolved in 1 mL of DMSO. The procedures that followed were the same as those applied to Gem-PPG100 as described above.

Gemcitabine loading efficiency was quantified through the application of reversed-phase high-performance liquid chromatography (HPLC).47 The analytical instrument utilized for this assessment was the Waters 1525 HPLC system, manufactured by Waters, USA. The chromatographic conditions were meticulously fine-tuned, employing a XSelect HSS C18 column characterized by dimensions of 4.6 mm × 250 mm and an inner diameter (i.d.) of 5 μm particles, all sourced from Waters, USA. Precise temperature control was maintained at 40 °C throughout the chromatographic procedure. The mobile phase composition consisted of two components: Mobile phase A was a 40 mmol/L ammonium acetate buffer with a pH of 5.5, while mobile phase B was composed of acetonitrile. An operational flow rate of 1.0 mL/min was utilized. Detection of the UV signal was achieved at 268 nm, employing 5-bromouracil as an internal standard for reference. The quantification of gemcitabine loading efficiency and loading capacity within the nanoparticles was accomplished via the application of eqs 1 and 2 as described below.481

2

Size, Zeta-Potential, and Surface Density Measurements

The size and zeta-potential of each micelle formulation were assessed via dynamic light scattering, employing the BI-200 SM instrument from Brookhaven Instruments, USA. Before the measurements, the micelle solution was allowed to equilibrate at room temperature for 1 h. The reported data represent the mean values obtained from three independent measurements, with the micelle concentration set at 1 mg/mL and a sample volume of 40 μL for each measurement.

The surface density was measured in two steps. First, the number of micelles was quantified in micelle solution (3 mg/mL), using a nanoparticle tracking analyzer (NanoSight NS300, Malvern Panalytical Ltd., England). The measurement was repeated three times. The track of nanoparticles in the field of vision was recorded; the quantity was calculated automatically by the software. Second, gemcitabine micelle solution was diluted by 10 mM Hepes buffer (pH 7.5) into nine concentrations, titrated with Ni2+ ion and pyrocatechol violet (PV), respectively. The absorbance value was measured on the ultraviolet–visible spectrophotometer (400–800 nm). The surface density of carboxyl and GCA on micelles were calculated by eqs 3 and 4 below.443

4

In Vitro Gemcitabine Release Study

In vitro gemcitabine release was quantified using a dialysis method as described in previous studies.49 Briefly, the micellar suspensions were dispersed in distilled water and subsequently introduced into dialysis membrane bags with a molecular weight cutoff of 14 kDa. These bags were securely sealed and then immersed in vials containing fresh release medium and specifically prepared PBS (20 mL) with 2% Tween80 at pH 7.4. Gemcitabine’s release from the micelles was executed using an air bath shaker set to 37 °C. Throughout a 120 h evaluation period, 3 mL of the outer solution was routinely withdrawn and replaced with fresh buffer at predefined time intervals. Gemcitabine concentrations in each of these samples (n = 3) were determined via HPLC, employing the aforementioned analytical method. The drug release experiments under simulated conditions mimicking intestinal fluid (pH 6.8) and gastric fluid (pH 1.2) were conducted in a similar manner.

Cellular Uptake Studies

Caco-2 cells were cultivated on polycarbonate filters within Transwell 24-well plates until they achieved a confluent monolayer with an 80% cell fusion rate. Prior to commencing the experimental procedures, the monolayers of Caco-2 cells underwent a meticulous washing process with PBS (pH 7.4). Following this, the monolayers were equilibrated with prewarmed PBS for a duration of 30 min, a procedure conducted at a controlled temperature of 37 °C within an incubator enriched with a 5% CO2 environment. In order to visualize the cellular uptake of the nanoparticles, the donor solutions, located at the apical side, were meticulously prepared. This involved the dilution of an aliquot of DiO-PPG60 and DiO-PP100 solution, each having a concentration of 100 nM, followed by removing the PBS. After subjecting the Caco-2 cell monolayers to various formulations for a period of 30 min, they were subjected to another cycle of PBS washing, following which the supporting membranes were excised from the insets. These membranes were then affixed onto microscope slides, subjected to staining with DAPI, and subsequently shielded with coverslips. The resulting slides were meticulously examined employing CLSM with the use of an FV1000 system (Olympus, Japan), while the quantification of DiO dye content was carried out utilizing flow cytometry facilitated by an LSRFortessa system (BD, USA), as previously documented.50

Transport Studies through the Caco-2 Cell Monolayer

Transwell inserts characterized by transepithelial electrical resistance (TEER) values within the range of 1000–1200 Ω × cm2 were employed. Caco-2 cell monolayers were subjected to two sequential PBS washes and subsequently immersed in prewarmed HBSS (pH 7.4). This incubation occurred for a duration of 30 min at 37 °C within an incubation chamber that maintained a controlled environment of 5% CO2. The donor solutions were methodically prepared by diluting an aliquot of DiO-PPG60 and DiO-PP100 solutions into HBSS to achieve a final DiO concentration of 45 mg/mL. At discrete time intervals, a volume of 200 μL from each acceptor sample was judiciously withdrawn, and an equivalent volume of fresh HBSS was promptly replenished within the acceptor compartment. Furthermore, the integrity of the cellular monolayer was assessed through the measurement of TEER values. The determination of the apparent Papp for DiO followed a well-defined procedure, utilizing eq 5 as outlined below. In this equation, dCr/dt characterizes the slope representing the linear relationship between the cumulative quantities of insulin DiO transported over time. A symbolizes the membrane area, measured in square centimeters (cm2), while C0 signifies the initial concentration of insulin within the donor compartment, in adherence to established protocols.515

ASBT-Mediated Endocytosis Studies

Following a 1 h incubation period with either PBS, DiO-PPG60, or DiO-PP100 micelles, the Caco-2 cell monolayers underwent a meticulous triple wash procedure employing PBS. Subsequently, the cells were firmly fixed with a 4% paraformaldehyde solution for a duration of 30 min. Following fixation, an immunofluorescent staining protocol targeting ASBT was meticulously executed. This procedure featured the application of an ASBT goat polyclonal antibody as the primary antibody, followed by the utilization of Cy3-labeled donkey anti-goat IgG as the secondary antibody. Once the staining process was completed, the supporting membranes were expertly excised from the Transwell inserts, mounted onto microscope slides, and subjected to an additional staining step involving DAPI. Finally, these slides were meticulously observed under CLSM to acquire detailed insights into ASBT localization and distribution.51

Cytotoxicity of Gemcitabine Micelles in BxPC-3 and Mia-Paca-2 Cancer Cells

Human pancreatic cancer cell lines BxPC-3 and Mia-Paca-2 were purchased from ATCC. Both cell lines were maintained under culture conditions, per guidelines, at 37 °C and 5% CO2. The tumor cells were routinely subcultured. The resultant cells growing in an exponential growth phase were harvested and counted for plating.

Antitumor efficacy was analyzed by a CTG assay. Cells were counted by hemocytometer with trypan blue staining. The cells were adjusted to proper density, and a 135 μL cell suspension was plated into the assay, followed by a 135 μL assay medium in the blank wells. The plates were incubated at 37 °C, 5% CO2, and 100% relative humidity overnight. The test particles (10× concentration of work concentration) were diluted, and the resulting 15 μL of diluted solution was added into wells. The assay plates then were replaced into the incubator and incubated for another 5 days. To calculate the relative light unit (RLU) values of cell viability at day 1 and day 5, 75 μL of CellTiter-Glo reagent (Promega, USA) was added to each well, the plates were gently shaken at room temperature for 10 min, and the luminescence was recorded on the 2104 EnVision plate reader (Envision, USA). The growth inhibition rate (GIR) value was calculated by eq 6 below.496

In Vivo Blood Gemcitabine Level and Bioavailability

Male Sprague–Dawley rats, with a mean weight of 200 ± 20 g, were procured from the Guangdong Medical Laboratory Animal Center (Guangdong, China). These animals were accommodated in a controlled environment maintained at a temperature of 25 °C and a humidity level of 50%. Furthermore, they were afforded ad libitum access to both standard rat chow and water resources. Ethical approval was obtained (approval number SH9H-2021-A766-SB) and issued by the ethics committee of Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine (Shanghai, China). All animal experiments complied with the National Institutes of Health, Guide for the Care and Use of Laboratory Animals (NIH publications No. 8023, revised 1978). Twelve animals were allocated randomly to form four groups (n = 3 per group). The first group was intravenously injected with Gem-PPG100 micelles; the second and third groups received either 60 or 100 nm Gem-PPG micelles by oral gavage. The fourth group was administered unmodified Gem-PP100 micelles orally. All experimental groups were administered a gemcitabine dosage of 10 mg/kg. Subsequently, blood samples were systematically collected at predefined time intervals with each sample volume measuring 0.2 mL and treated with heparin sodium as an anticoagulant. These collected samples were then promptly preserved at a temperature of −80 °C to maintain their integrity for subsequent analysis. Gemcitabine in the plasma was extracted, 1 mL of organic solvent (methanol–acetonitrile = 1:9) was added, and the resulting solution was vortexed for 2 min and centrifuged at 6000 rpm for 5 min. The supernatant was lyophilized, redissolved with 200 μL of ammonium acetate buffer (pH 5.5), vortexed for 2 min, and centrifuged at 6000 r/min for 5 min. A 50 μL amount of supernatant was collected and determined by HPLC, as described above. The pharmacokinetic parameters were meticulously ascertained by utilizing the software Origin8.5, developed by OriginLab in the United States. These parameters encompassed the maximum plasma concentration (Cmax), the time at which Cmax was achieved (Tmax), the half-life (T1/2), and the total area under the curve (AUC) calculated from the pharmacokinetic profile generated by the software. Furthermore, the absolute oral bioavailability (F) for each formulation was calculated employing eq 7, as stipulated below,527

Biodistribution Study

Male Sprague–Dawley rats, weighing 200–225 g, were housed at 25 °C and 50% humidity, with free access to food and water. Ethical approval was obtained (approval number SH9H-2021-A766-SB) and issued by the ethics committee of Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine (Shanghai, China). Six were allocated randomly to form two groups (n = 3 per group). One group was intravenously injected with free gemcitabine hydrochloride solution, while the other received Gem-PPG60 micelles by oral gavage. Both groups received a gemcitabine dose of 10 mg/kg. The target organs (heart, liver, spleen, lung, kidney, and pancreas) from the euthanized rats were collected 8 h postadministration. Blood samples were collected and determined by HPLC, as described above. The organs were collected, quickly frozen (3–5 min), and crushed by tissue homogenizer to yield a powder formulation. The extraction and determination of gemcitabine content of each organ were followed by the same plasma method described above. The degrees of drug distribution in the different organs were evaluated by the gemcitabine concentration ratio of tissues/plasma.53

Noninvasive Optical Imaging of Tumor-Bearing Mice

To confirm the absorption and noninvasive imaging of drug micelles, DiR was used instead of gemcitabine.54 Ethical approval was obtained (approval number SH9H-2021-A766-SB) and issued by the ethics committee of Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine (Shanghai, China). Mice, orally administered with saline and 60 nm DiR-PPG micelles (DiR = 200 μg/kg), respectively, were anesthetized by a respiratory oxygen–isoflurane mixture. Images were acquired with an in vivo fluorescence imaging system as follows. The animals were positioned on the imaging platform, and images were taken at time intervals after the oral administration of the DiR-PPG micelles. The main organs and tumors from the euthanized mice were immediately collected, and the fluorescence was observed to assess the nanomicelle distribution. Fluorescence and white-light imaging were selected. The excitation and emission wavelengths of DiR were 720 and 790 nm, respectively, and the exposure time was 30 s. Image analysis was performed using an IVIS Spectrum CT imaging system (PerkinElmer, USA).

In Vivo Pharmacodynamic Study

BxPC-3 tumor cells were cultured, and a total volume of 200 μL of RPMI 1640 medium (Gibco, USA) containing BxPC-3 cells at a concentration of 1 × 107 cells per 0.2 mL was collected. Subsequently, this cell suspension was injected subcutaneously into the right cervical back region of BALB/c mice.55 Ethical approval was obtained (approval number SH9H-2021-A766-SB) and issued by the ethics committee of Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine (Shanghai, China). To evaluate therapeutic effects, the BALB/c mice were allocated randomly to form three groups (n = 5 per group). Over a period of 33 days, free gemcitabine solution (60 mg/kg, BIW) was administered by intraperitoneal injection, and saline and Gem-PPG60 micelles (30 mg/kg, BIW) were administered orally at 2–3 p.m., 1 h prior to the first meal of the day. Measurements of the tumor size and body weight were taken twice weekly throughout the study duration. On the 33rd day, the experiment was concluded, the mice were humanely euthanized, and tumor specimens were collected from each group for further analysis. The tumor growth inhibition value (TGI) was calculated for each group using eq 8 below,56 where T33 is the average tumor volume of the treatment group on the 33rd day, T0 is the average tumor volume of the treatment group on the day of grouping, V33 is the average tumor volume of the control group on the 33rd day, and V0 is the average tumor volume of the control group on the day of grouping.8

Hematology, Blood Chemistry, and Histology Study

To determine any incidental toxicity from administration of the drug solution and the drug-loaded micelles, several preliminary toxicity assessments were conducted.57 After 33 days, three mice were randomly selected from each of the saline, drug solution, and drug-loaded micelle groups and euthanized, and the heart, liver, spleen, lung, kidney, pancreas, stomach, ileum, colon, and tumor were excised with necropsies. Blood samples (600 μL) were collected from the jugular vein, and 150 μL was analyzed by hematology analyzer (HEMAVET950, USA) to determine white blood cell, red blood cell, and platelet levels. Remaining blood samples were centrifuged (3000g, 25 °C, 15 min), and the levels of alanine aminotransferase (ALT) (L-Type ALT IFCC, Wako, Japan), aspartate transaminase (AST) (L-Type AST IFCC, Wako, Japan), and creatinine (CRE) (L-Type Creatinine M, Wako, Japan) in the plasma were quantified with an assay kit. The harvested organs were promptly immersed in a 4% buffered formaldehyde solution for a duration of 48 h to ensure proper fixation. Subsequently, these preserved organs were meticulously sectioned into coronal slices measuring 4 μm in thickness by utilizing a precision microtome. These tissue sections underwent a comprehensive evaluation through hematoxylin and eosin staining, which was subsequently followed by a thorough pathological examination. Immunostaining of the tumor specimens was executed through a series of meticulous steps, commencing with a 10 min incubation in a 3% hydrogen peroxide solution. This was followed by thorough rinsing with Tris buffer saline and then further incubation with rabbit anti-human Ki-67 antibody (9129, CST, USA) at a dilution ratio of 1:300 for a duration of 30 min. To evaluate the Ki-67 index, the percentage of Ki-67-positive cells relative to the total cell count was calculated in three distinct hotspots (×20) observed across three separate slices from each experimental group.

Statistical Analysis

Statistical data analyses were performed using Microsoft Excel 2020 (Microsoft, USA) and GraphPad (Insight Partners, USA). Differences between the values were assessed using Student’s t-tests.

Data Availability Statement

All pertinent data supporting the crucial discoveries of this research endeavor are accessible within the primary article and the Supporting Information.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.3c04793.Figures showing 1H NMR spectra of GCA and COOH–PEG5k–PLGA10k, surface density measurements of 100 nm gemcitabine-loaded micelles, surface density measurements of 60 nm gemcitabine-loaded micelles, the TEM image and DLS data of the gemcitabine-loaded micelles, in vitro nanoparticle transport efficacy using the Caco-2 model, in vitro antitumor efficacy study of gemcitabine solution and unloaded micelles, in vitro antitumor efficacy study of gemcitabine-loaded micelles on U87 glioma cancer cells, permeability assay of gemcitabine micelles in PAMPA, immunohistochemistry of tumors by Ki-67, histology studies (PDF)

Supplementary Material

nn3c04793_si_001.pdf

Author Contributions

W.Z., D.G., and M.Y. contributed equally to this work. W.Z., conceptualization, methodology, formal analysis, investigation, validation, data analysis, writing – original draft, supervision, funding acquisition. D.G., investigation, validation, data analysis. M.Y., investigation, methodology, data analysis, supervision. Z.Z., investigation, validation. T.J., writing – review & editing, supervision, project administration, funding acquisition.

The authors declare no competing financial interest.

Acknowledgments

We are grateful for the financial support from the National Natural Science Foundation of China (91959125 and 81972336 to T.J.), the International Cooperation Research Projects of Shanghai Science and Technology Commission (19410740300 to T.J.), the Cross Disciplinary Research Projects of Ninth People’s Hospital, Shanghai Jiao Tong University, School of Medicine (JYJC202106 to T.J.), the China Postdoctoral Science Foundation (2021M702181 to W.Z.), and Young Elite Scientists Sponsorship Program by CAST (2022QNRC001 to M.Y.). We are grateful for the technical support of pharmacokinetic and pharmacodynamic experiments from WuXi AppTec.
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