A dual chemodrug-loaded hyaluronan nanogel for differentiation induction therapy of refractory AML via disrupting lysosomal homeostasis

Shilin Xu; Tao Wang; Xuechun Hu; Hong Deng; Yiyi Zhang; Lei Xu; Yang Zeng; Jia Yu; Weiqi Zhang; Lin Wang; Haiyan Xu

doi:10.1126/sciadv.ado3923

. 2025 Mar 28;11(13):eado3923. doi: 10.1126/sciadv.ado3923

A dual chemodrug-loaded hyaluronan nanogel for differentiation induction therapy of refractory AML via disrupting lysosomal homeostasis

Shilin Xu ¹, Tao Wang ¹, Xuechun Hu ¹, Hong Deng ^1,², Yiyi Zhang ^1,², Lei Xu ³, Yang Zeng ³, Jia Yu ⁴, Weiqi Zhang ^1,^2,^*, Lin Wang ^4,^*, Haiyan Xu ^1,^*

PMCID: PMC11952094 PMID: 40153509

Abstract

Relapsed/refractory acute myeloid leukemia (rrAML) is a malignant blood cancer with an extremely poor prognosis, largely ascribed to the drug-resistant leukemia stem cells (LSCs). Most patients suffer from a risk of difficult-to-cure as well as severe systemic toxicity when receiving standard chemotherapies. As hyaluronic acid (HA) is a specific ligand of CD44 highly expressed by LSCs, we had HA self-assembled with cisplatin and daunorubicin to form a dual chemodrug nanogel (HA/Cis/Dau) to afford the targeted therapeutic interventions of rrAML. HA/Cis/Dau displayed an extra therapeutic function of inducing the granulocyte-monocyte differentiation in CD44⁺ rrAML cells, an rrAML mouse model, and primary blasts isolated from patients with AML. Unlike free drugs directly diffusing and killing rrAML cells, HA/Cis/Dau transported the drugs into lysosomes, causing lysosomal membrane permeabilization, ROS accumulation, and thus a metabolic reprogramming of the rrAML cells. Moreover, HA/Cis/Dau was featured with alleviated side effects, ease of preparation, and cost effectiveness, therefore holding great promises for the targeted treatment of rrAML.

A HA/Cis/Dau nanogel enables chemo-differentiation therapy of AML via inducing lysosomal membrane permeabilization.

INTRODUCTION

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by clonal expansion of myeloid blasts or progranulocytes with impaired differentiation (1). In the past three decades, the global AML incidence was almost doubled, and meanwhile, around 100,000 of AML-related death was reported recently (2). The standard treatments for AML, which mainly rely on the intensive chemotherapy and hematopoietic stem cell transplantation, remained unchanged for more than 40 years (1, 3). Despite the progress of the current treatment strategies for AML achieved in recent years, the prognosis of most patients with refractory or relapsed AML (rrAML), who fail to achieve a complete remission or will relapse later after achieving it, is still unsatisfactory (4, 5).

When patients with rrAML receive standard intensive chemotherapies, a fraction of leukemia stem cells (LSCs) would survive and thus largely attributable to the high rate of relapse and drug resistance (6). Moreover, the nontargeting chemotherapeutics indiscriminately attack normal cells of the patients with AML, leading to severe systemic toxicity. Besides the intensive chemotherapies, differentiation induction is considered an ideal alternative approach for treating AML, which, in principle, can induce leukemic cells/LSCs to differentiate into normal-looking and nonmalignant cells and thus cure patients with relatively low toxic effects (7). However, despite all-trans retinoic acid and arsenic trioxide displayed the notable differentiation induction effects and are applied clinically in the treatment of acute promyelocytic leukemia, the differentiation therapy showed limited efficacy in other multiple subtypes of AML (8, 9). Considering the dismal prognosis and the absence of satisfactory treatment options, there is still an unmet need to improve the cure rate as well as the tolerance of chemotherapeutic interventions, especially for those patients with rrAML. Specific targeting and eliminating of LSCs represent a promising approach to develop safer and more efficient therapeutics to improve patients’ remission rates and quality of life.

The cell receptor CD44 that is overexpressed on a variety of leukemia cells has been suggested as a LSC marker (10, 11). While CD44 is highly correlated with tumor progression/metastasis, drug resistance, and poor prognosis, it also provides an ideal target to guide the cancer-selective drug delivery (12, 13). The natural polysaccharide, hyaluronic acid (HA), is a specific ligand of CD44 and has been widely used as biomaterials for different clinical applications including pharmaceutical excipient in exploiting the CD44-targeted delivery for AML treatments (14–16). Among various therapeutics delivered by HA-based carriers, cisplatin (Cis) that functions as a DNA alkylating agent has unique performance of coordinating with HA’s carboxyl groups, providing a powerful strategy to construct various HA-based nanocarriers with ease (17–19). We previously developed the platinum coordination between Cis and HA to form multidrug nanocarriers that could be efficiently taken up by CD44⁺ tumor cells, thus enabling a facile and cost-effective formulation for CD44-targeted delivery (16, 17, 20). Following the CD44-mediated endocytosis of HA, it was noted that HA-based nanocarriers delivered the encapsulated drugs into endolysosomes, an intracellular location that is routinely observed for most of cancer nanomedicine (21, 22). The biomedical values underlying the lysosomal trapping of nanocarriers have been largely ignored, although extensive efforts are focusing on promoting the lysosomal escape to facilitate the intracellular drug delivery (20, 23, 24). It is widely documented that lysosomal homeostasis is physiologically important in cancer biology, especially that lysosomes are suggested as an ideal therapeutic target for cancers including AML (25, 26).

From the view of lysosomal homeostasis versus nanodelivery system, in this work, we developed a self-assembled HA nanogel (referred to as HA/Cis/Dau) to afford the CD44-targeted codelivery of Cis and daunorubicin (Dau), a first-line anthracycline drug clinically used in AML therapy. The targeted therapeutic effects and lysosomal perturbation of the HA/Cis/Dau were investigated with CD44⁺ rrAML cells and an rrAML mouse model of AML1-ETO (AE) & CKIT^D816V and validated in primary blasts isolated from patients with AML. We showed that HA/Cis/Dau acquired an additional therapeutic function of inducing the rrAML cells to differentiate into granulocyte-monocytes through altering the trafficking of delivered drugs in rrAML cells. The free Cis or Dau directly diffused, accessed nucleus, and killed rrAML cells. However, the HA/Cis/Dau located in the lysosomes caused lysosomal membrane permeabilization (LMP), accumulation of intracellular reactive oxygen species (ROS), and subsequently the induction of rrAML cell differentiation through metabolism reprogramming. Moreover, HA/Cis/Dau substantially diminished the systemic toxicity of the chemotherapeutic drugs due to the CD44-targeted ability and the induced differentiation. These results highlighted the translational potential of the HA/Cis/Dau nanogel by combining chemotherapy and differentiation therapy for rrAML treatments.

RESULTS AND DISCUSSION

The HA/Cis/Dau nanogel was prepared on the basis of the cooperative self-assembly between HA, Cis, and Dau (Fig. 1A), which did not require chemical alteration of HA and thus featured with a facile and cost-effective preparation. The multiple noncovalent interactions, mainly the platinum coordination and electrostatic interaction between Cis, Dau, and HA, were believed to mediate the formation of HA/Cis/Dau nanogels as we previously demonstrated (17, 18). In HA/Cis/Dau, Cis not only acted as a cross-linker through the coordination with HA carboxyl groups to hold the nanogel structure but also functioned as a chemotherapeutic after its release. The HA/Cis/Dau presented as nanospheres with a diameter of around 38.5 nm as observed by the atomic force microscopy (AFM) and transmission electron microscopy (TEM) (Fig. 1 and fig. S1). The nanogel was negatively charged with a hydrodynamic diameter of 143.8 nm in aqueous solution [polydispersity index (PDI) = 0.2] (Fig. 1D and table S1). The diameter increase of HA/Cis/Dau in water in reference to the TEM size was ascribed to the adsorption of water molecules into the nanogel because HA is highly hydrophilic. The elemental mapping demonstrated the presence of platinum within the HA/Cis/Dau nanogel, which was also confirmed with the x-ray photoelectron spectroscopy (XPS) (Fig. 1, E to G). Successful loading of Dau in the nanogel was clearly reflected in absorption, fluorescence, and Fourier transform infrared spectroscopy (FTIR) spectra (Fig. 1, E and F, and fig. S2). For HA/Cis/Dau, the COO⁻ peak of Cis around 1618 cm⁻¹ of HA was red shifted to 1643 cm⁻¹ and the absence of NH₂− peak of Cis is ~800 cm⁻¹ (Fig. 1I), which are presumably due to the platinum coordination with carboxyl groups and the resultant distortion of amine groups within this nanogel (27). Compared with HA only, HA/Cis/Dau demonstrated an infrared band that corresponds to the C═O peak of Dau (~1713 cm⁻¹), evidencing its successful encapsulation (28, 29). In addition, the red shift of the absorption peak and fluorescent quench of Dau collectively suggested a Dau aggregation within the HA/Cis/Dau, which has also been observed by us and others previously (24, 30, 31). The self-aggregation of Dau together with its interaction with HA led to a Dau encapsulation and loading efficiency of 87.5 and 9.86%, respectively (table S2). The encapsulation efficiency of Cis was 38.7%, corresponding to a loading efficiency of 26.2%. Both Cis and Dau demonstrated a controlled release behavior in phosphate-buffered saline (PBS) buffer (Fig. 1G). In contrast to Cis release, release of Dau was slower and less which could be ascribed to the chemical-physical differences between these two chemodrugs. Nevertheless, both drugs were expected to be efficiently released under the biological settings, because HA is readily degraded by hyaluronidase (HAase) that is overexpressed in various cancers (32, 33). The HAase responsiveness was verified by the accelerated release of both Cis and Dau when the HA/Cis/Dau was coincubated with HAase (fig. S3). After the drug release in PBS buffer, the hydrodynamic diameter of HA/Cis/Dau was slightly increased because of a partial removal of Cis as cross-linker (fig. S4, A and B). In addition, no aggregation of HA/Cis/Dau was observed in the dynamic light scattering (DLS) size distribution graph after 48 hours of incubation in PBS containing 10% fetal bovine serum (fig. S4, C and D), which could be due to the excellent hydrophilicity and antifouling properties of HA. These data collectively suggested that HA/Cis/Dau could be well dispersed with the contact of serum proteins and thus suitable for the in vivo injection. Furthermore, the HA/Cis/Dau size was stable for up to 21 weeks when stored in aqueous solution at 4°C (fig. S5), indicating the excellent stability of current HA/Cis/Dau nanogels.

Taking advantages of Dau’s intrinsic fluorescence, the intracellular uptake of the HA/Cis/Dau was evaluated using the fluorescence confocal microscopy and flow cytometry [fluorescence-activated cell sorting (FACS)]. The representative rrAML cell line Kasumi-1 and the chronic myeloid leukemia cell line K562 that have distinct CD44 expression levels were used as model cells (Fig. 2, A and B). As both were evaluated by microscopy and FACS, the HA/Cis/Dau demonstrated more Dau fluorescence in Kasumi-1 cells that have a higher CD44 expression when compared with K562 cells (Fig. 2, D and E, and fig. S6A). In addition, a similar uptake trend of Cis in these two cell lines was confirmed through a direct cellular platinum quantification using the inductively coupled plasma mass spectrometry (ICP-MS) (fig. S6B). With the presence of extra free HA in culture medium, the competition assay clearly reduced HA/Cis/Dau fluorescence in Kasumi-1 cells (Fig. 2C), supporting the CD44-targeting potential of HA-based carriers for AML cells as reported before (11, 12). It was noted that the free Dau strongly stained the nucleus in both cell lines, while HA/Cis/Dau signals were relatively weak and mainly located in lysosomes (Fig. 2D). This dimmer fluorescence in the case of HA/Cis/Dau-treated cells could be resulted from the quenched Dau fluorescence within the nanogel. Unlike free Dau to directly penetrate the cell membrane and locate to the nucleus, HA was endocytosed and subsequently metabolized in endolysosomes (19, 21), which well explained the lysosomal location of HA/Cis/Dau fluorescence. The HA/Cis/Dau demonstrated a clearly dose-dependent cytotoxicity toward Kasumi-1 cells, while its efficiency was weaker than the Cis + Dau (Fig. 2F and fig. S7). The compromised cytotoxicity of HA/Cis/Dau could be ascribed to the slowed release and the altered intracellular localization of the encapsulated drugs, which were commonly observed for nanoparticle formulations in vitro (34, 35). Nevertheless, the HA/Cis/Dau still maintained its payload’s chemotoxicity as evidenced by the effects to induce cell apoptosis, while the HA only failed to cause noticeable toxicity (Fig. 2G).

Fig. 2. — The CD44 level on Kasumi-1 (A) and K562 (B) cells. (C) The cellular uptake of HA/Cis/Dau in the presence of extra free HA. Kasumi-1 cells were incubated with HA/Cis/Dau for 15 min. MFI, mean fluorescence intensity. (D and E) Confocal microscopy. Kasumi-1 (D) and K562 (E) cells were labeled by 4′,6-diamidino-2-phenylindole (DAPI) and LysoTracker to visualize nucleus and lysosome, respectively. Colocalization of LysoTracker and Dau fluorescence in cells (along the dashed lines) was analyzed by ImageJ. Scale bar, 10 μm. (F) Cell viability of Kasumi-1 cells after 24-hour incubation (n = 4). (G) Apoptosis assay. Cells were stained by annexin V and 7-AAD. **P < 0.01, ***P < 0.001, and ****P < 0.0001. Con, control.

As the pharmacodynamics of the nanomedicine plays a pivotal role in determining the therapeutic efficacy (36, 37), Cis and Dau in the peripheral blood (PB) were evaluated in healthy mice after the systemic administration of HA/Cis/Dau. The blood circulation times of both Cis and Dau in HA/Cis/Dau clearly outperform the Cis + Dau group (Fig. 3, A and B). The longer retention of HA/Cis/Dau in blood circulation would maintain an effective therapeutic window of drugs for rrAML treatment. Thus, the therapeutic efficacy was then evaluated using the AE & CKIT^D816V mouse model with rrAML features (38–40). Mice were irradiated by x-ray to destroy the hematopoietic ability, and then green fluorescent protein (GFP)–tagged rrAML cells were intravenously inoculated (Fig. 3C). The GFP tag allowed a facile and effective monitoring of the disease progress by counting the fluorescent cells (rrAML cells) in the PB. When the rrAML cells reach around 10% of the blood cells, a typical leukemia cell infiltration rate for rrAML model, the mice were administered with saline, HA only, Cis + Dau, and HA/Cis/Dau with Cis and Dau dose set at 3.3 and 1.25 mg/kg, respectively. Through six repeated intravenous injection every 3 days, the control mice all died after 27 days due to the infiltration of rrAML cells in the main organs. Notably, HA/Cis/Dau induced a longer survival of mice as 75% of mice survived after 40 days, while only 20% of mice were alive and a median survival time of 31 days was observed in the case of Cis + Dau (Fig. 3D). In addition, the HA vehicle group did not show a therapeutic effect at the same HA dose that was used in the HA/Cis/Dau. Consequently, the elongated survival of mice in Fig. 3D was ascribed to the nanoparticulate formulation of HA/Cis/Dau. To verify this, the disease burden was evaluated by analyzing the rrAML (GFP-positive) cells in the main organs after four doses of different formulations. Both the HA/Cis/Dau and Cis + Dau treatments led to a notable decrease of rrAML cells in the PB, bone marrow (BM), spleen, liver, and lung due to the chemotoxicity of Cis and Dau in vivo (Fig. 3, E to I). When compared with HA/Cis/Dau, the GFP-positive cells (rrAML) were even less in the spleen and BM for those mice treated with Cis + Dau. This discrepancy between rrAML cell infiltration and mouse survival further stimulated us to investigate the therapeutic benefits of HA/Cis/Dau nanogel with a focus on AML cell differentiation. The rrAML cells from the mouse spleen and BM, which are the main organs suffering from leukemia burden, were isolated directly and incubated with HA/Cis/Dau and Cis + Dau of equivalent drug content (Fig. 3K), and then the differentiation marker, including CD14 and CD11b that correspond to the granulocyte and monocyte differentiation, was evaluated, respectively (41). Compared with Cis + Dau, the HA/Cis/Dau could mediate a significant up-regulation of both CD14 and CD11b in rrAML cells from the spleen (fig. S8) and BM (Fig. 3, L and M). The elevated differentiation markers of rrAML cells were further affirmed by costaining the GFP together with CD14 or CD11b of BM in situ. As shown in Fig. 3N, while the Cis + Dau mainly reduced the GFP-positive (rrAML) cells, HA/Cis/Dau could clearly up-regulate both the CD14 and CD11b levels of the rrAML cells. This evidence suggested that more rrAML cells in the HA/Cis/Dau-treated mice could be converted to granulocyte- and monocyte-like cells, possibly through the differentiation-inducing effect of the HA/Cis/Dau nanogel. Although the differentiated cells still retained the GFP tag, they were less malignant, which well explains that more GFP⁺ cells were present in the spleen and BM, while the corresponding survival was longer for HA/Cis/Dau-treated mice in refence to those treated with Cis + Dau.

After the therapeutic intervention of the AE & CKIT^D816V mice (Fig. 3C), it was also noted that the spleens of Cis + Dau–treated mice were even smaller than those of healthy mice (Fig. 3J and fig. S9), which implied a nonspecific toxicity of Cis + Dau. The toxicity of Cis + Dau in the AML mice was also evidenced on the basis of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) and histochemical staining of the main organs. Compared to the control, HA, and HA/Cis/Dau groups, severe pathological changes were observed in the spleen, kidney, and liver in the Cis + Dau–treated mice (fig. S10). This was further verified by the elevated cell death in the collected organs, as reflected by TUNEL staining (fig. S11). Further immunohistochemical staining of cytokeratin-18 (CK-18) and α-smooth muscle actin (α-SMA), two biomarkers that reflect liver and kidney damage, respectively, confirmed the nonspecific toxicity of Cis + Dau in multiple organs (fig. S12). This may also contribute to the shortened survival time of the AML mice compared with the HA/Cis/Dau group. The side effects were notorious for combined chemotherapy; thus, the safety profile of the HA/Cis/Dau nanogel in refence to Cis + Dau was further comprehensively evaluated using the healthy mice. After the same treatments that were used in the therapeutic experiments, blood cells and main organs of the healthy mice were analyzed. The Cis + Dau significantly induced a decrease in red blood cells (RBCs), white blood cells (WBCs), platelet (PLT), and hemoglobin (HGB) (Fig. 4, A to D). In the HA/Cis/Dau-treated group, most cell numbers in the blood were similar to those of control and HA vehicle, except that there was a clear decrease in PLT; however, this value was comparable to that of the Cis + Dau group. In addition, the biochemical analysis including liver injury markers aspartate aminotransferase (AST), total protein (TP), and albumin (ALB) and kidney injury markers globulin (G), blood urea nitrogen (BUN), and uric acid (UA) showed that HA/Cis/Dau treatment did not exhibit any significant difference compared with the control group, affirming an acceptable safety of HA/Cis/Dau nanogel (fig. S13). In hematoxylin and eosin (H&E) staining, pathological features in the main organs of healthy mice were also observed for Cis + Dau, such as the destruction of tissue architecture in the BM, kidney, and heart, etc. (labeled by black arrows), while less changes were found for the HA/Cis/Dau group, referring to the control and HA groups (fig. S14). The main concern of Cis for leukemia treatment is the myelosuppression as well as the nephrotoxicity (42), while the cardiotoxicity was common for Dau as a typical anthracycline drug (43); thus, the histological staining of the main organs was further evaluated. The platinated DNA adducts (Cis-DNA) that directly account for the Cis toxicity were stained in the BM and kidney using the immunofluorescence. Compared with the Cis + Dau group, there were less Cis-DNA adducts both in the BM and kidney for the HA/Cis/Dau group, strongly suggesting that the Cis toxicity could be relieved by HA/Cis/Dau (Fig. 4, E and F). Furthermore, the Masson and Sirius Red staining of the heart tissue suggested that the HA/Cis/Dau nanogel attenuated the chemodrug-induced cardiac fibrosis changes in contrast with the Cis + Dau group both in AML mice and healthy mice (Fig. 4, G and H). In general, the toxicity related to Cis and Dau could be efficiently reduced by forming the HA/Cis/Dau nanogel. Moreover, HA/Cis/Dau could potentially provide a new therapeutic option for rrAML by the integration of both chemotherapy and differentiation therapy.

Fig. 4. — (A to D) Count measurements of WBCs, RBCs, PLTs, and HGB in the PB (n = 3). (E and F) Cis-DNA staining of the BM (E) and kidney (F) in healthy mice. The red fluorescence detected the platinated DNA (Cis-DNA adducts). (G and H) After the same treatment regimen, comparison of myocardial morphology by H&E, Masson, and Sirius Red staining in AML mice (G) and healthy mice (H). Scale bars, 20 μm. *P < 0.05 and **P < 0.01. Con, control.

To probe the underlying mechanism of the superior therapeutic efficacy of HA/Cis/Dau in vivo, the chemo-differentiation effects were firstly confirmed by the Wright-Giemsa staining of the treated Kasumi-1 cells (Fig. 5A). The Cis + Dau generated a staining pattern full of cell debris, suggesting the induction of cell apoptosis as verified in apoptosis assay (Fig. 2G), which is also evident from the fragmented nucleus as stained by 4′,6-diamidino-2-phenylindole (DAPI) (Fig. 5B). While the HA/Cis/Dau led to a larger cell size with a heterogeneous nucleus in Wright-Giemsa staining as well as a clearly enlarged nucleus in DAPI staining (~2-fold increase in an area) compared with control (Fig. 5, A and B), thus confirming the granulocyte-monocyte differentiation (44, 45). The HA/Cis/Dau also resulted in a notable up-regulation of differentiation markers including CD14 and CD11b as determined by FACS and immunofluorescence analysis (Fig. 5, B and D, and fig. S15, A and B). The granulocyte-monocyte differentiation induction of HA/Cis/Dau was further affirmed by the real-time polymerase chain reaction (PCR) analysis of the mRNA expression of CEBPA and SPI1 (fig. S16), the typical transcription factors controlling the granulocyte differentiation, and the KLF4 that critically regulates the monocyte differentiation (46, 47). The above morphological and biochemical evidences collectively supported that HA/Cis/Dau gained an increased capability to induce the differentiation of AML cells compared to the Cis + Dau. To probe whether this differentiation induction was specific for the HA/Cis/Dau nanogel, its effect on CD11b and CD14 expression was compared with the HA/Cis/Rb nanogel that coloaded with Cis and rhodamine B (Rb; a model drug with minimal toxicity) (48) and HA nanogels containing a single drug (HA/Cis, HA/Dau, or HA/Rb) with the involved drug concentrations set equally (fig. S17). While the HA/Rb failed to induce the differentiation of the Kasumi-1 cells, HA/Cis and HA/Dau demonstrated an increased level of both CD11b and CD14, suggesting that the payload in these HA nanogels determined their differentiation induction outcome. Notably, HA/Cis/Dau induced the most up-regulations of both CD11b and CD14 among all the tested nanogels, affirming that the observed strongest granulocyte-monocyte differentiation relied on its coencapsulation of both Cis and Dau. While the intrinsic chemotoxicity of the delivered Cis and Dau could kill the leukemia cells directly, the HA/Cis/Dau also mediated an effective differentiation therapy and thus collectively contributed to an improved survival of the AML mice as observed above. Both platinum-based (e.g., Cis) and anthracycline drugs (e.g., Dau) have demonstrated limited potential to stimulate the differentiation of malignant cells. However, the underlying mechanism remains unclear (7, 49, 50). The pharmacological action sites of both Cis and Dau are mainly nucleus as well documented. Nevertheless, the HA/Cis/Dau mainly delivered the payloads into lysosomes as observed by confocal microscopy (Fig. 2B). The sequestration of toxic drugs in lysosomes could generate lysosomal stress, causing LMP and redox imbalance; thus, the lysosomal integrity was evaluated by immunofluorescence staining of galectin-3, a marker for LMP, and the Magic Red assay, an indicator for lysosomal leakage (19, 51). HA/Cis/Dau treatment obviously caused LMP as indicated by the galectin-3 puncta (fig. S18), which was comparable to the lysosomal damage generated by L-leucyl-L-leucine methyl ester (LLoMe), a commonly used dipeptide that induces LMP (fig. S19). Leakage of the representative lysosomal protein cathepsin B was confirmed on the basis of diffusive Magic Red fluorescence in cytosol in the case of HA/Cis/Dau-treated cells either compared with Cis + Dau or HA treatment (Fig. 5C), affirming that the nanogel produced LMP. As the LMP was actively involved in ROS homeostasis in cells (52), the intracellular ROS was evaluated by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. It was found that the HA/Cis/Dau led to an increased intracellular ROS in a dose-dependent manner, which differed from that of HA and Cis + Dau (Fig. 5E). Furthermore, the proteome profiler array of cell stress–related proteins confirmed that HA/Cis/Dau markedly elevated the cellular levels of Bcl-2, HSP60, and phosphorylated P53 (Fig. 5F), which are key proteins that strongly associated with ROS signaling. Pretreating the cells with antioxidant N-acetyl-l-cysteine (NAC) could not prevent the LMP process caused by HA/Cis/Dau (fig. S20); however, it could significantly reduce the expression of differentiation markers (Fig. 5D and fig. S15). When the HA/Cis/Dau-induced LMP was inhibited by the U18666A (fig. S18), a small molecule that modifying cholesterol transport and thus stabilizing the lysosome (53), clear down-regulation of intracellular ROS and differentiation markers was observed in Kasumi-1 cells (figs. S21 and S22). While the homeostasis of intracellular ROS was highly complicated, these results strongly suggested that HA/Cis/Dau caused LMP and subsequently the ROS accumulation that played a pivotal role in promoting the differentiation of AML cells as previously reported (41, 54). The HA/Cis/Dau nanogels that enhanced the ROS level in a concentration-dependent manner were also observed in rrAML cells isolated from the spleen or BM of AE & CKIT^D816V mouse (fig. S23), implying that HA/Cis/Dau has the ability to modulate ROS levels in vivo.

Fig. 5. — (A) Wright-Giemsa staining of cells after 5-day treatment [Cis (0.45 μg/ml) and Dau (0.175 μg/ml)]. (B) Immunofluorescence staining of CD14 and the statistical data of the nucleus areas stained by DAPI. Scale bar, 20 μm. (C) Cathepsin B evaluation by a Magic Red assay kit and the relative integrated density of Magic Red in cytoplasm. Scale bar, 10 μm. (D) FACS analysis of the CD14 expression with or without NAC pretreatment (n = 3). (E) Relative ROS in Kasumi-1 cells incubated with Cis + Dau and HA/Cis/Dau with or without NAC pretreatment (n = 3). (F) Proteome profiler array. “*,” “#,” or “&” P < 0.05; “**,” “##,” or “&&” P < 0.01; ***P < 0.001; ****P < 0.0001. Con, control.

Lysosome is the essential catabolic organelle of the cells, and it has been proposed as a key target organelle for AML therapy (25). To further probe the mechanism underlying the differentiation induction effects of HA/Cis/Dau, metabolomic analysis was performed considering that the metabolome can accurately reflect the functional and phenotype changes in cells. An untargeted metabolomic analysis was conducted on Kasumi-1 cells treated with various formulations. As shown in Fig. 6, while the change of 154 confirmed metabolites was similar for HA only in reference to the control, a distinct pattern of metabolites was observed between HA/Cis/Dau and Cis + Dau groups. The most notable differences between Cis + Dau and HA/Cis/Dau were found in amino acid, lipid, as well as the redox-related metabolisms (Fig. 6A). A higher abundance of numerous amino acids was found in AML cells treated with HA/Cis/Dau. For example, the amino acids such as leucine, serine, threonine, phenylalanine, aspartate, and asparagine, as well as glutamine, glutamate, glutathione, proline, and glycine, were all present in higher concentrations in HA/Cis/Dau treatment samples (Fig. 6, B and C, and fig. S24). These amino acids could serve as precursors or indirect participants to generate several important metabolites including pyruvate, lactate, α-ketoglutarate, and malate, which were closely linked to a process such as glycolysis, tricarboxylic acid cycle, or glutaminolysis (55). Furthermore, levels of the glycerophospholipids (glycerophosphoglycerol) and long-chain acylcarnitines (hydroxybutyrylcarnitine and phosphodimethylethanolamine) involved in the glycerophospholipid metabolism pathway or fatty acid oxidation were obviously elevated by HA/Cis/Dau treatment (Fig. 6C). HA/Cis/Dau had a considerable impact on the redox metabolites, particularly nicotinamide adenine dinucleotide (oxidized form) (NAD⁺) and reduced form of NAD⁺ (NADH). As shown in Fig. 6B, cells treated with HA/Cis/Dau have an accumulation level of NAD⁺ and NADH, with an increased ratio of NAD⁺/NADH. The administration of NAC, an ROS scavenger, could reduce intracellular NAD⁺ level in HA/Cis/Dau-treated cells and normalize the NAD⁺/NADH ratio (Fig. 6D). NAD⁺ level is highly correlated to the cellular and metabolic signaling regulation (56, 57), and its role in inducing cell differentiation was further confirmed by inhibiting the NAD synthesis. Kasumi-1 cells were cotreated with FK866, an inhibitor for nicotinamide phosphoribosyltransferase (NAMPT) that accounts for the NAD synthesis (58). In contrast to the cases of HA- or Cis + Dau–treated cells, FK866 effectively suppressed the expression of both the CD11b and CD14 differentiation markers that were stimulated by the HA/Cis/Dau (Fig. 6E). It has been reported that NAD⁺ induces differentiation in promyelocytic leukemia by directly supplementing NAMPT to promote NAD⁺ production in cells (58). While, here, we demonstrated that HA/Cis/Dau could induce NAD⁺ accumulation, which plays a crucial role in promoting the granulocyte-monocyte differentiation of AML cells. It was also noted that the differentiation markers up-regulated by the HA/Cis/Dau could not be fully blocked by the NAD inhibition (Fig. 6E). This suggested that there might be additional metabolites contributing to the differentiation of leukemia cells induced by HA/Cis/Dau. HA/Cis/Dau treatment also increased many other metabolites, including pyruvate, lactate, α-ketoglutarate, succinate, threonine, leucine, asparagine, hydroxybutyrylcarnitine, and phosphodimethylethanolamine. Conversely, Cis + Dau treatment showed a suppressive effect on these metabolites, which likely accounts for the varied therapeutic response observed in rrAML cells. Comprehensive investigations will be needed to fully identify the key metabolites as well as unveil the underlying mechanism for the differentiation therapy enabled by the HA/Cis/Dau. Nevertheless, our findings strongly supported that HA/Cis/Dau could induce LMP and ROS accumulation, rewiring of metabolism such as NAD-related redox metabolism in the rrAML cells, and ultimately culmination in the cell differentiation in addition to the chemotoxicity exerted by Cis and Dau.

Fig. 6. — (A) Heatmap of metabolite expression in Kasumi-1 cells treated with different formulations. Color code for higher abundance is red, and color code for lower abundance is blue. (B and C) The representative metabolic variation induced by HA/Cis/Dau in Kasumi-1 cells (n = 3). (D) The intracellular NAD⁺ or NADH was determined using NAD⁺/NADH assay kit. (E) FACS analysis of the CD14 and CD11b expression with or without FK866 coincubation (n = 3). *P < 0.05, **P < 0.01. UTP, uridine triphosphate; UDP, uridine diphosphate; ATP, adenosine triphosphate; GDP, guanosine diphosphate; dCMP, 2′-deoxycytidine 5′-monophosphate; FAD, flavin adenine dinucleotide; CoA, coenzyme A; Con, control.

To further verify the efficacy of HA/Cis/Dau treatment that was more clinically relevant, BM aspirates of four patients diagnosed with different AML subtypes were acquired with informed consent (table S3). The primary blasts were incubated with HA/Cis/Dau or Cis + Dau for 24 hours [Cis (0.45 μg/ml) and Dau (0.175 μg/ml)]. It was shown that HA/Cis/Dau could significantly increase the CD11b and CD14 expression on the blasts from patient 1, which was in well accordance with that observed in the Kasumi-1 cell line and the leukemia mouse model (Fig. 7, A and B). Among the other three patients, HA/Cis/Dau could enhance the levels of either CD11b or CD14, while in patients 2 and 4, the Cis + Dau only led to a partial elevation of CD11b or CD14 (Fig. 7C and fig. S25). As there were only a small number of primary blasts available from the patients, the morphological and apoptosis analysis was tested in samples only from the first two patients (Fig. 7, B and D). The HA/Cis/Dau induced both apoptosis and differentiation, as observed using Wright-Giemsa staining, while Cis + Dau treatment primarily caused chemotoxicity (cell apoptosis). As summarized in table S3, the varied differentiation-inducing effect (the CD11b and CD14 levels) could be resulted from the individual heterogenicity as well as the different leukemia subtype of patients. Abnormalities in the genetic and epigenetic composition of AML cells have the potential to disrupt normal differentiation pathways, leading to a state of low differentiation (59). The differentiation therapy has been proved as an effective treatment modality for AML, a fact amply demonstrated by the success of all-trans retinoic acid. Here, HA/Cis/Dau has been observed to induce differentiation in the rrAML mice and extend their survival, suggesting that it would also provide clinical benefits through differentiation induction and thus potentially offer an additional option for patients with rrAMLs who are unresponsive to conventional chemotherapy. Considering the heterogenicity of AML, future translational application of current HA/Cis/Dau may also require optimization based on a larger number of patient samples and a detailed phenotyping of patients with AML.

Fig. 7. — (A) FACS analysis of the CD11b and CD14 expression on the primary AML blasts from patient 1 (M2a, n = 3). (B) Wright-Giemsa staining of the primary AML blasts from patient 1. (C) FACS analysis of the CD11b and CD14 expression on the primary AML blasts from patient 2 (M5, n = 3). (D) Apoptosis assay of the primary AML blasts from patient 2. *P < 0.05, **P < 0.01, and ****P < 0.0001. Con, control.

To summarize, the current work provided a facile and practicable formulation of HA/Cis/Dau nanogel for rrAML therapy through the self-assembly between HA, Cis, and Dau. Compared with Cis + Dau, HA/Cis/Dau nanogel could be selectively delivered into the lysosomes in CD44-overexpressed rrAML cells, which switched the trafficking of chemotherapeutics from entering cells by passive diffusion to CD44-mediated endocytosis and meanwhile efficiently reduced the systemic toxic effects. The mechanism studies suggested that the HA/Cis/Dau not only maintained the chemotoxicity of the encapsulated drugs but also acquired an additional function of inducing the granulocyte-monocyte differentiation of rrAML cells through causing LMP, which led to the accumulation of intracellular ROS and subsequently the metabolic reprogramming (e.g., NAD⁺ accumulation) of the rrAML cells (Fig. 8). Collectively, the differentiation induction effects of HA/Cis/Dau were confirmed using CD44⁺ rrAML cells, an rrAML mouse model of AE & CKIT^D816V, and the primary blast isolated from the patients, suggesting the translational potential of this rrAML treatment regimen. Our strategy successfully provided a proof of concept to use the self-assembled nanogel to fully unleash the therapeutic potential of clinically used chemodrugs for rrAML. Future efforts could focus on elucidating the molecular mechanism of HA/Cis/Dau’s role to optimize the differentiation-inducing effects, which would help further improve the therapeutic outcome of rrAML.

Fig. 8. — Free drugs (Cis + Dau) penetrate the cell membrane, access the nucleus, and cause cell apoptosis directly, while the HA/Cis/Dau delivered the payloads into lysosomes, inducing LMP, ROS generation, and metabolic reprogramming of AML cells, which collectively induced the cell differentiation other than the chemotoxicity inherent to Cis and Dau. TCA, tricarboxylic acid; Con, control.

MATERIALS AND METHODS

Materials

HA with a molecular weight of 1000 kDa was purchased from Lifecore Biomedical (Chaska, MN, USA). Cis (#479306), Rb (#R6626), and HAase (#H3506) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dau hydrochloride was purchased from TCI Chemicals (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan), and o-phenylenediamine (OPDA) was purchased from Macklin Chemicals (Macklin Co. Ltd., Shanghai, China). U18666A was purchased from TargetMol (#T17190) (Boston, MA, USA).

Cell culture

Kasumi-1 cells were obtained from the Institute of Hematology and Blood Diseases Hospital (Tianjin, China). Cells were cultured in modified RPMI 1640 medium (HyClone, GE Healthcare Life Sciences, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), penicillin (100 U/ml), and streptomycin (100 U/ml) (HyClone) at 37°C in a humidified atmosphere of 5% CO₂.

Preparation and characterization of nanogels

The HA/Cis/Dau nanogel was prepared on the basis of a previous protocol (17, 19). Briefly, Cis (15 mg/ml) in double-distilled water (ddH₂O) was preheated at 90°C to allow full dissolution. Then, 200 μl of Cis was mixed with an equal volume of Dau (2.5 mg/ml in ddH₂O) before adding 800 μl of HA solution (5 mg/ml in ddH₂O). The mixture was incubated at 90°C for 3.5 hours and then cooled on ice for 5 min. After that, the solution was dialyzed against 1 liter of ddH₂O for 1 day to remove the unloaded drugs. Dialysis water was also collected to quantify the Dau content based on its fluorescence intensity, according to the Dau standard curve. The HA/Cis and HA/Cis/Rb nanogels were prepared by heating the mixture of HA and Cis or HA, Cis, and Rb solution for 2 hours using the same protocol described above. For the HA/Dau and HA/Rb nanogel preparation, the as-prepared HA/Cis/Dau and HA/Cis/Rb nanogels (2-hour heating) were subjected to the dialysis in PBS (0.1 M, pH 7.4) at 37°C for 3 days to remove the Cis as described elsewhere (33). Cis in the nanogel was quantified using the OPDA method. The acquired HA/Cis/Dau nanogel was diluted in 9% NaCl solution at the rate of 100-fold. An equal volume of OPDA dissolved in N,N′-dimethylformamide (1.2 mg/ml) was added and mixed thoroughly. After 10 min of heating at 100°C, absorbance at 705 was recorded using the Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA). The Cis content in nanogel was calculated using the Cis standard dissolved in ddH₂O. The DLS analysis of HA/Cis/Dau was conducted on a Zetasizer Nano ZS90 machine (Malvern Instruments, Malvern, UK). Briefly, 50 μl of HA/Cis/Dau was diluted with 1 ml of ddH₂O and subjected to DLS analysis with three repeated measurements. Then, 200 μl more of HA/Cis/Dau was added in the above solution for the zeta potential determination using the DLS machine. For fluorescent spectra, HA/Cis/Dau and free Dau (10 μg/ml) were scanned on the plate reader with excitation set at 480 nm (emission, 500 to 750 nm). The absorption spectra of HA/Cis/Dau and free Dau solution (20 μg/ml) in cuvette were measured using the UH5300 spectrophotometer (Hitachi Ltd., Tokyo, Japan) with the wavelength set from 250 to 700 nm. HA or Cis of the same concentration with that in HA/Cis/Dau was included as control. The FTIR was conducted using a Nicolet iS20 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The powder of HA, Cis, Dau, and lyophilized HA/Cis/Dau was palletized in potassium bromide (KBr) pellets and subjected to FTIR measurements in the range of 4000 to 400 cm⁻¹. For XPS analysis of HA/Cis/Dau, the lyophilized nanogel was subjected to the survey and high-resolution XPS spectrum measurements using the ESCALAB 250Xi XPS instrument (Thermo Fisher Scientific, Waltham, MA, USA). The morphology of HA/Cis/Dau nanogel was then observed using the transmission electron microscope (JEM 2100F, JEOL, Tokyo, Japan) and the atomic force microscope (Dimension Icon, Bruker Corporation, USA). Briefly, 20 μl was mixed with 30 μl of pure water, and then 5 μl of the diluted nanogel was deposited on the copper grid or mica sheet for TEM and AFM measurements, respectively. For TEM observation, the sample on the grid was stained with 1.5% uranaly acetate for 2 min before the observation. To map the element distribution in nanogel, the HA/Cis/Dau dropped on the grid was imaged on a JEOL JEM-F200 machine (JEOL, Tokyo, Japan) equipped with an X-Max 80 T energy dispersive spectroscopy (EDS) detector, and the C, N, O, Cl, and Pt element signals were collected on the basis of the TEM observation.

Drug release experiment

One milliliter of HA/Cis/Dau solution was sealed in the dialysis tube (molecular weight cut-off, 3.5 kDa) and dialyzed in 80 ml of PBS (0.1 M, pH 7.4). The drug release experiment was conducted at 37°C on a shaker with a rotation speed set at 100 rpm. At the predetermined time points, 1 ml of PBS was collected, and meanwhile, an equal volume of fresh PBS was replenished. To evaluate the HAase-responsive release of HA/Cis/Dau, the nanogel was dispersed in PBS buffer with or without HAase (50 U/ml, pH 5). Then, the mixture was sealed in a dialysis tube and subjected to the drug release analysis. The Cis and Dau content in the collected PBS was quantified on the basis of the OPDA and Dau fluorescence as described above. The accumulated drug release curve was calculated on the basis of the determined Cis and Dau content.

Cell viability assay

Kasumi-1 cells were seeded onto 96-well plates at a density of 5 × 10⁵/ml. The cell viability was analyzed after incubation with Cis + Dau and HA/Cis/Dau at different concentrations (HA, 0.3 to 15 μg/ml; Cis, 0.09 to 4.5 μg/ml; Dau, 0.035 to 1.75 μg/ml) for 24 and 48 hours. After incubation, 10 μl of Cell Counting Kit-8 (Dojindo Laboratories, Japan) was added to each well. Cells were incubated at 37°C for another 1.5 hours. Then, the optical density values were read at 450 and 630 nm using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). Untreated cells were considered as control.

Cellular uptake assay

Kasumi-1 or K562 cells were incubated with Cis + Dau and HA/Cis/Dau (HA, 15 μg/ml; Cis, 4.5 μg/ml; Dau, 1.75 μg/ml) for 2.5 hours at 37°C. Then, cells were stained with LysoTracker Deep Red (Thermo Fisher Scientific, Waltham, MA, USA) and coincubated for another 0.5 hours at 37°C. After incubation, cells were washed with PBS and then fixed with 4% paraformaldehyde for 0.5 hours at room temperature. Nuclei were stained with DAPI at 4°C overnight. Red fluorescence of Dau, green fluorescence of LysoTracker, and blue fluorescence of DAPI were acquired by confocal laser scanning microscopy (Olympus FV1000, Center Valley, PA); colocalization of LysoTracker and Dau in cells treated with different drugs was analyzed by ImageJ software. Meanwhile, cells were treated with the same conditions as above, and the fluorescence of Dau in cells was detected using a C6 Accuri flow cytometer. For Cis quantification, the treated cells were harvested and subjected to nitric acid digestion, and then the Pt content was determined using a 7800 ICP-MS instrument (Agilent Technologies, USA). For the competition assay, Kasumi-1 cells were treated with HA/Cis/Dau (HA, 15 μg/ml; Cis, 4.5 μg/ml; Dau, 1.75 μg/ml) in the absence or presence of excess free HA for 15 min. Then, cells were collected and detected using a C6 Accuri flow cytometer.

Cell apoptosis assay

Kasumi-1 cells of 5 × 10⁵/ml were seeded onto six-well plates and incubated with HA, Cis + Dau, and HA/Cis/Dau (HA, 15 μg/ml; Cis, 4.5 μg/ml; Dau, 1.75 μg/ml) for 24 hours. Cells were collected and washed with PBS. Then, resuspended cells in the binding buffer were stained with fluorescein isothiocyanate (FITC)–annexin V and 7-amino-actinomycin D (7-AAD; Elabscience Biotechnology Co. Ltd., China) at room temperature for 15 min. A total of 1 × 10⁴ cells were collected and subjected to flow cytometry, and acquired data were analyzed by FlowJo 7.6 software.

Cell differentiation assay in vitro

Kasumi-1 cells of 5 × 10⁵/ml were seeded onto 6-cm cell culture dishes and incubated with HA, Cis + Dau, and HA/Cis/Dau (HA, 1.5 μg/ml; Cis, 0.45 μg/ml; Dau, 0.175 μg/ml) for 5 days. On the third day of incubation, the medium was changed, and the drug was readded to stimulate for 2 days. Then, cells were collected and washed with PBS, followed by stained with FITC-conjugated antihuman CD11b and CD14 antibodies (BioLegend, USA). The labeled cells were detected using a C6 Accuri flow cytometer. For the rescue experiment, cells were pretreated with 5 mM NAC (Sigma-Aldrich, USA) for 1 hour, and then the cells were incubated with the above drugs for 5 days. Later, cells were incubated with the differentiation markers and then detected using a flow cytometer. In addition, cells were coincubated with 10 nM FK866 (MedChemExpress, USA) and drugs for 3 days or pretreated with 5 μM U18666A for 1 hour and then treated with the above drugs as indicated. After that, cells were treated with the differentiation markers and then detected using a flow cytometer. Furthermore, Kasumi-1 cells were incubated with HA/Cis, HA/Dau, HA/Rb, HA/Cis/Dau, and HA/Cis/Rb nanogels with the involved drugs fixed to the equivalent concentrations. After 3-day incubation, the treated cells were then stained with indicated markers and detected using a flow cytometer. In addition, after an incubation for 5 days, Kasumi-1 cells were collected, stained with FITC-conjugated antihuman CD14 and allophycocyanin (APC)–conjugated antihuman CD11b antibodies (BioLegend, USA), and then fixed with 4% paraformaldehyde for 0.5 hours at room temperature. Nuclei were stained with DAPI. Images were taken by confocal laser scanning microscopy at excitation wavelengths of 633 nm for APC, 488 nm for FITC, and 405 nm for DAPI. The nucleus areas from at least 100 cells were measured via ImageJ software and analyzed by SPSS 21.0. Cell samples were dried in air before stained with Wright-Giemsa for 40 to 50 s and then added buffer washing for 7 min. The light microscope (Olympus BX53, Japan) was used to observe images. Cells were treated with the above drugs for 3 days, and then the total RNA was extracted using TRIzol reagent (Life Technologies Corporation, USA) and reverse-transcribed to cDNA. The gene expression related to cell differentiation was examined by quantitative real-time PCR using TB Green Premix Ex Taq (Takara Bio, Japan). All primers used in the present study were synthesized from Sangon Biotech (Shanghai, China) and shown in table S4.

Cathepsin B assay

Kasumi-1 cells were incubated with LLoMe (Cayman Chemical, Ann Arbor, USA), HA, Cis + Dau, and HA/Cis/Dau (HA, 1.5 μg/ml; Cis, 0.45 μg/ml; Dau, 0.175 μg/ml) with or without NAC pretreatment. After 24-hour incubation, cells were followed by a Magic Red Cathepsin B assay kit according to the manufacturer’s protocol (Abcam, UK). Images were acquired by confocal laser scanning microscopy.

Galectin-3 staining

Kasumi-1 cells were incubated with LLoMe, HA, Cis + Dau, HA/Cis/Dau (HA, 1.5 μg/ml; Cis, 0.45 μg/ml; Dau, 0.175 μg/ml), and HA/Cis/Dau in the presence of 5 μM U18666A. After 24-hour incubation, cells were fixed with 4% paraformaldehyde for 0.5 hours and 0.5% Triton X-100 for another 20 min at room temperature. Cells were incubated with the galectin-3 antibody (Santa Cruz Biotechnology, USA) overnight at 4°C. Then, cells were labeled with APC goat anti-rat immunoglobulin G1 antibody (BioLegend, USA) for 1 hour. Images were acquired by confocal laser scanning microscopy.

ROS detection

Kasumi-1 cells were incubated with HA, Cis + Dau, and HA/Cis/Dau at different concentrations (HA, 1.5 to 6 μg/ml; Cis, 0.45 to 1.8 μg/ml; Dau, 0.175 to 0.7 μg/ml) for 24 hours. Then, cells were washed with PBS and incubated in 10 μM DCFH-DA (Merck KGaA, Germany) for 0.5 hours at 37°C. After incubation, cells were washed with PBS and analyzed using a C6 Accuri flow cytometer. For the rescue experiment, cells were pretreated with 5 mM NAC for 1 hour, and then the cells were incubated with the above drugs for 24 hours. Kasumi-1 cells were also pretreated with 5 μM U18666A for 1 hour, and then the cells were incubated with HA, Cis + Dau, and HA/Cis/Dau (HA, 1.5 μg/ml; Cis, 0.45 μg/ml; Dau, 0.175 μg/ml) for 24 hours. After that, cells were incubated with the DCFH-DA in the same manner and then detected using a flow cytometer. AML cells were isolated from the spleen or the BM and incubated with the same concentrations of drugs in Kasumi-1 cells. Later, cells were incubated with the Cellular ROS Assay Kit (Deep Red, Abcam, UK) at 37°C for 0.5 hours. Afterward, cells were analyzed by a C6 Accuri flow cytometer using the FL4-A channel.

Proteome profiler array

Kasumi-1 cells of 5 × 10⁵/ml were incubated with HA, Cis + Dau, and HA/Cis/Dau (HA, 1.5 μg/ml; Cis, 0.45 μg/ml; Dau, 0.175 μg/ml) for 24 hours. Cells were collected and washed with PBS. Then, the Human Cell Stress Array Kit (R&D Systems, USA) was used to detect the differences among different treatment groups. The technical steps were strictly followed by the instructions. Furthermore, the relative expression levels of cell stress related proteins were measured by ImageJ software.

Cell metabolism

Kasumi-1 cells were treated with HA, Cis + Dau, and HA/Cis/Dau (HA, 1.5 μg/ml; Cis, 0.45 μg/ml; Dau, 0.175 μg/ml) for 24 hours. Medium was aspirated, and metabolism was quenched with 80% methanol as the extraction buffer. Cells were placed on ice for 10 min and then centrifuged in a benchtop microfuge at the maximum speed for 30 min at 4°C. The supernatant was transferred to liquid chromatography–mass spectrometry vials (keep on ice) for analysis. To measure metabolites in cells, an orbitrap mass spectrometer (Orbitrap Exploris 480; Thermo Fisher Scientific) was coupled to a Vanquish Ultra-high performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific) with electrospray ionization and a scan range of mass/charge ratio from 70 to 1000, with a 120,000 resolution. A gradient of solvent A (95:5 water:acetonitrile containing 20 mM ammonium acetate and 20 mM ammonium hydroxide, pH 9.45) and solvent B (acetonitrile) was used for LC separation on an XBridge BEH Amide column (2.1150 mm, 2.5-m particle size; Waters). The rate of flow was 150 μl/min. The LC gradient was as follows: 0 min, 90% B; 2 min, 90% B; 3 min, 75%; 7 min, 75% B; 8 min, 70% B, 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 14 min, 25% B; 16 min, 0% B, 21 min, 0% B; 21 min, 90% B; and 25 min, 90% B. The injection volume was 8 μl, and the temperature of the autosampler was set to 4°C. Metabolite peaks were picked with signal to noise (S/N) at S/N > 3.

NAD⁺/NADH assay

Kasumi-1 cells were treated with HA, Cis + Dau, and HA/Cis/Dau (HA, 1.5 μg/ml; Cis, 0.45 μg/ml; Dau, 0.175 μg/ml) in the presence of NAC or not for 24 hours. Then, the content of NAD⁺ and NADH was detected using a NAD⁺/NADH assay kit with the WST-8 method (Beyotime, China) according to the manufacturer’s instructions.

Treatment in AE & CKIT^D816V mice

Six-week-old female C57 mice were maintained in the Experimental Animal Center at the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China) under specific pathogen–free conditions. All the animal experiments reported herein were carried out in accordance with the approved guideline and approved by the committee on the Animal Care and Use of Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College (#ACUC-A02-2022-004). Animals were acclimatized to laboratory conditions for 1 week before experiments.

Survival experiment

The procedure of AE & CKIT^D816V mouse model establishment was described in previous studies (38–40). Briefly, splenic cells from the moribund AE & CKIT^D816V mice were isolated and injected intravenously into the sublethally irradiated (450 cGy) secondary recipient C57 mice. The AE & CKIT^D816V cells were also labeled with GFP. After 9 days, mice were randomly divided into four groups: Control (stroke-physiological saline solution, n = 14), HA (n = 14), Cis + Dau (n = 14), and HA/Cis/Dau (HA, 11.5 mg/kg; Cis, 3.3 mg/kg; Dau, 1.25 mg/kg; n = 13). The administration was given by intravenous injection every 3 days for a total of six times. The overall survival of AML mice was recorded every day until death.

Pharmacodynamic experiment

In the pharmacodynamic experiment, the mice of all groups (Control, HA, Cis + Dau, and HA/Cis/Dau; n = 4) were euthanized 24 hours after the fourth administration. The percentage of AE & CKIT^D816V cells in the PB, BM, spleen, liver, and lung was analyzed using a C6 Accuri flow cytometer. The counts of WBCs, RBCs, and PLTs from the PB were detected using the auto hematology analyzer (Mindray Bio-Medical Electronics Co. Ltd., China). The main organs including the heart, liver, spleen, lung, and kidney were also fixed with 4% paraformaldehyde and embedded in paraffin for further histological staining analysis. For TUNEL staining and immunohistochemistry, the fixed tissue sections from AML mice were treated using the TUNEL apoptosis detection kit (Servicebio, China) following the manufacturer’s instructions. Meanwhile, the liver and kidney slides from the AML mice were stained by CK-18 and α-SMA antibodies (Servicebio, China), respectively. After the incubation with horseradish peroxidase–labeled secondary antibodies following a standard protocol, the samples were then examined under a microscope.

Cell differentiation in vivo

The AE & CKIT^D816V cells isolated from the BM and spleen in the moribund AML mice were incubated with phycoerythrin (PE)–conjugated anti-mouse CD11b and CD14 antibodies (BioLegend, USA). The labeled cells were determined using the C6 Accuri flow cytometer. In addition, BM tissues were routinely processed and stained with H&E, GFP, CD11b, or CD14 (Servicebio, China).

Pharmacokinetic experiment in vivo

Female C57 mice were randomly divided into two groups (n = 3) and injected intravenously with Cis + Dau and HA/Cis/Dau at doses of 3.3 mg/kg Cis, 1.25 mg/kg Dau, and 11.5 mg/kg HA. Approximately 500 μl of blood samples was collected at 3 min, 10 min, 0.5, 1, 2, 4, 8, 24, and 48 hours postinjection and then centrifuged at 3500 rpm for 15 min at 4°C to harvest serum samples, and aqua regia was added to digest overnight. An atomic absorption spectrometer (Hitachi High-Tech Science, Japan) was used to detect the platinum (Pt) content in all serum samples. For pharmacokinetics of Dau, female C57 mice were randomly divided into two groups (n = 4) and injected intravenously with Cis + Dau and HA/Cis/Dau at doses of 16.5 mg/kg Cis, 6.25 mg/kg Dau, and 57.5 mg/kg HA. Ten microliters of blood sample was collected at 3 min, 10 min, 0.5, 1, 2, 4, 8, 24, and 48 hours postinjection and mixed with 10 μl of anticoagulant. Then, the fluorescence values of Dau in the collected blood samples were read at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using the microplate reader. The blood samples from healthy mice not treated with any drugs were served as the blank control group.

Toxicity evaluation of HA/Cis/Dau

Six- to 8-week-old female healthy C57 mice were randomly divided into Control, HA, Cis + Dau, and HA/Cis/Dau groups (n = 3), and the administration regimen was the same as AE & CKIT^D816V mice. All mice were euthanized after the sixth intravenous injection. Organs from the C57 healthy mice were fixed with 4% paraformaldehyde and embedded in paraffin.

Blood cell analysis

The number of WBCs, RBCs, PLTs, and HGB in the PB was analyzed using the auto hematology analyzer. For blood biochemical analysis, serum samples were acquired by centrifugation at 3500 rpm for 15 min at 4°C. AST, TP, ALB, G, BUN, and UA were determined using a biochemical automatic analyzer (Beckman Coulter, USA).

Histological and immunofluorescent staining

BM and kidney sections were first deparaffinized and rehydrated. Then, the permeabilize working solution was added to the sections to cover the objective tissue, and then the sections were incubated at room temperature for 20 min. After the slices were slightly dried, buffer was added to the tissues in the circle, and the buffer was incubated at room temperature for 10 min. Then, the slices were incubated with the anti-Cis–modified DNA antibody (Abcam, UK) at 4°C overnight. Last, cells were stained with PE-conjugated secondary antibodies (Abcam, UK) and DAPI solution and then photographed with an EVOS M7000 microscope (Thermo Fisher Scientific, Waltham, MA, USA). DAPI emitted blue light at an ultraviolet excitation wavelength of 380 nm and an emission wavelength of 420 nm; PE had an excitation wavelength of 480 nm and an emission wavelength of 560 nm and emitted red light. For cardiac toxicity assessment, all tissues sections were stained with H&E in routine ways, besides that the heart sections were extra stained with Masson and Sirius red. Images were observed using the light microscope.

Patient samples

Patients with newly diagnosed AML provided informed consent under the Fifth Medical Center of Chinese PLA General Hospital–approved protocol (KY-2022-4-19-1) and were the source of primary AML blasts for in vitro resimulation experiments. The primary blasts from the BM aspirates of patients with AML were isolated by lymphocyte isolation media (TBDScience, China) and immediately used in experiments. Cells were treated with HA, Cis + Dau, and HA/Cis/Dau (HA, 1.5 μg/ml; Cis, 0.45 μg/ml; Dau, 0.175 μg/ml) for 24 hours. Then, the cell differentiation detection, cell apoptosis assay, and Wright-Giemsa staining were performed as same as described above.

Statistical analysis

All data were expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) or Student’s t test was used for the results. Statistical analysis was performed in GraphPad Prism 8.0 software. Survival functions were compared by the log-rank test. P value < 0.05 was considered to be statistically significant.

Acknowledgments

Funding: This work was supported by the National Key R&D Program of China (2022YFA1205803 to H.X. and W.Z.) and (2022YFA1106300 to Y.Z. and L.W.), Haihe Laboratory of Cell Ecosystem Innovation Fund (22HHXBSS00040 to H.X., and W.Z.), National Natural Science Foundation of China (82200260 to S.X.), the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2021-RC350-008 to L.W.), State Key Laboratory Special Fund (2060204 to L.W. and W.Z.), Young Talent Foundation of PLA General Hospital (2019-YQPY-002 to Y.Z.), the Open Project Program of the State Key Laboratory of Proteomics (SKLPK-0202006 to L.X.), and Beijing Nova Program (Z201100006820110 to W.Z.) of Beijing Municipal Science and Technology Commission.

Author contributions: S.X. performed conceptualization, investigation, methodology, validation, formal analysis, data curation, visualization, funding acquisition, writing—original draft, and writing—review and editing. T.W. performed investigation and writing—review and editing. X.H. performed investigation and validation. H.D. performed investigation and validation. Y.Z. performed investigation and validation. L.X. contributed to resources and funding acquisition. Y.Z. performed investigation, visualization, resources, funding acquisition, and writing—review and editing. J.Y. contributed to writing—review and editing. W.Z. performed conceptualization, investigation, methodology, validation, formal analysis, data curation, visualization, supervision, funding acquisition, writing—original draft, and writing—review and editing. L.W. performed investigation, methodology, validation, formal analysis, data curation, visualization, resources, funding acquisition, and writing—review and editing. H.X. performed conceptualization, methodology, resources, project administration, supervision, funding acquisition, writing—original draft, and writing—review and editing. All authors agreed to submit the manuscript, read and approved the final draft, and took full responsibility of its content.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

Supplementary Materials

This PDF file includes:

Figs. S1 to S25

Tables S1 to S4

sciadv.ado3923_sm.pdf^{(4.2MB, pdf)}

REFERENCES AND NOTES

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figs. S1 to S25

Tables S1 to S4

sciadv.ado3923_sm.pdf^{(4.2MB, pdf)}

[R1] 1.Newell L. F., Cook R. J., Advances in acute myeloid leukemia. BMJ 375, n2026 (2021). [DOI] [PubMed] [Google Scholar]

[R2] 2.Yi M., Li A., Zhou L., Chu Q., Song Y., Wu K., The global burden and attributable risk factor analysis of acute myeloid leukemia in 195 countries and territories from 1990 to 2017: Estimates based on the global burden of disease study 2017. J. Hematol. Oncol. 13, 72 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R3] 3.Yang X., Wang J., Precision therapy for acute myeloid leukemia. J. Hematol. Oncol. 11, 3 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] 4.Thol F., Heuser M., Treatment for relapsed/refractory acute myeloid leukemia. Hema 5, e572 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R5] 5.Montesinos P., Bergua J., Infante J., Esteve J., Guimaraes J. E., Sierra J., Sanz M. Á., Update on management and progress of novel therapeutics for R/R AML: An Iberian expert panel consensus. Ann. Hematol. 98, 2467–2483 (2019). [DOI] [PubMed] [Google Scholar]

[R6] 6.Vetrie D., Helgason G. V., Copland M., The leukaemia stem cell: Similarities, differences and clinical prospects in CML and AML. Nat. Rev. Cancer 20, 158–173 (2020). [DOI] [PubMed] [Google Scholar]

[R7] 7.Thé H., Differentiation therapy revisited. Nat. Rev. Cancer 18, 117–127 (2018). [DOI] [PubMed] [Google Scholar]

[R8] 8.Abdel-Aziz A. K., Advances in acute myeloid leukemia differentiation therapy: A critical review. Biochem. Pharmacol. 215, 115709 (2023). [DOI] [PubMed] [Google Scholar]

[R9] 9.Madan V., Koeffler H. P., Differentiation therapy of myeloid leukemia: Four decades of development. Haematologica 106, 26–38 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] 10.Zöller M., CD44: Can a cancer-initiating cell profit from an abundantly expressed molecule? Nat. Rev. Cancer 11, 254–267 (2011). [DOI] [PubMed] [Google Scholar]

[R11] 11.Jin L., Hope K. J., Zhai Q., Smadja-Joffe F., Dick J. E., Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat. Med. 12, 1167–1174 (2006). [DOI] [PubMed] [Google Scholar]

[R12] 12.Qiu J., Cheng R., Zhang J., Sun H. L., Deng C., Meng F. H., Zhong Z. Y., Glutathione-sensitive hyaluronic acid-mercaptopurine prodrug linked via carbonyl vinyl sulfide: A robust and CD44-targeted nanomedicine for leukemia. Biomacromolecules 18, 3207–3214 (2017). [DOI] [PubMed] [Google Scholar]

[R13] 13.Mattheolabakis G., Milane L., Singh A., Amiji M. M., Hyaluronic acid targeting of CD44 for cancer therapy: From receptor biology to nanomedicine. J. Drug Target. 23, 605–618 (2015). [DOI] [PubMed] [Google Scholar]

[R14] 14.Kim H., Jeong H., Han S., Beack S., Hwang B. W., Shin M., Oh S. S., Hahn S. K., Hyaluronate and its derivatives for customized biomedical applications. Biomaterials 123, 155–171 (2017). [DOI] [PubMed] [Google Scholar]

[R15] 15.Harrer D., Armengol E. S., Friedl J. D., Jalil A., Jelkmann M., Leichner C., Laffleur F., Is hyaluronic acid the perfect excipient for the pharmaceutical need? Int. J. Pharm. 601, 120589 (2021). [DOI] [PubMed] [Google Scholar]

[R16] 16.Bai H., Wang T., Kong F., Zhang M., Li Z., Zhuang L., Ma M., Liu F., Wang C., Xu H., Gu N., Zhang Y., CXCR4 and CD44 dual-targeted Prussian blue nanosystem with daunorubicin loaded for acute myeloid leukemia therapy. Chem. Eng. J. 405, 126891 (2021). [Google Scholar]

[R17] 17.Zhang W., Tung C.-H., Cisplatin cross-linked multifunctional nanodrugplexes for combination therapy. ACS Appl. Mater. Interfaces 9, 8547–8555 (2017). [DOI] [PubMed] [Google Scholar]

[R18] 18.Zhang Y., Wang F., Li M., Yu Z., Qi R., Ding J., Zhang Z., Chen X., Self-stabilized hyaluronate nanogel for intracellular codelivery of doxorubicin and cisplatin to osteosarcoma. Adv. Sci. 5, 1700821 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R19] 19.Zhang W., Du B., Gao M., Tung C.-H., A hybrid nanogel to preserve lysosome integrity for fluorescence imaging. ACS Nano 15, 16442–16451 (2021). [DOI] [PubMed] [Google Scholar]

[R20] 20.Gao M. H., Deng H., Zhang Y. Y., Wang H. M., Liu R. M., Hou W., Zhang W., Hyaluronan nanogel co-loaded with chloroquine to enhance intracellular cisplatin delivery through lysosomal permeabilization and lysophagy inhibition. Carbohydr. Polym. 323, 121415 (2024). [DOI] [PubMed] [Google Scholar]

[R21] 21.Knudson W., Chow G., Knudson C. B., CD44-mediated uptake and degradation of hyaluronan. Matrix Biol. 21, 15–23 (2002). [DOI] [PubMed] [Google Scholar]

[R22] 22.Rennick J. J., Johnston A. P., Parton R. G., Key principles and methods for studying the endocytosis of biological and nanoparticle therapeutics. Nat. Nanotechnol. 16, 266–276 (2021). [DOI] [PubMed] [Google Scholar]

[R23] 23.Qiu C., Xia F., Zhang J., Shi Q., Meng Y., Wang C., Pang H., Gu L., Xu C., Guo Q., Advanced strategies for overcoming endosomal/lysosomal barrier in nanodrug delivery. Research 6, 0148 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] 24.Zhang W., Tung C.-H., Lysosome enlargement enhanced photochemotherapy using a multifunctional nanogel. ACS Appl. Mater. Interfaces 10, 4343–4348 (2018). [DOI] [PubMed] [Google Scholar]

[R25] 25.Rafiq S., McKenna S. L., Muller S., Tschan M. P., Humbert M., Lysosomes in acute myeloid leukemia: Potential therapeutic targets? Leukemia 35, 2759–2770 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R26] 26.Chauhan N., Patro B. S., Emerging roles of lysosome homeostasis (repair, lysophagy and biogenesis) in cancer progression and therapy. Cancer Lett. 584, 216599 (2023). [DOI] [PubMed] [Google Scholar]

[R27] 27.Guo H., Wang H., Gao M., Deng H., Zhang Y., Gong J., Zhang W., Harnessing the CD44-targeted delivery of self-assembled hyaluronan nanogel to reverse the antagonism between cisplatin and gefitinib in NSCLC cancer therapy. Carbohydr. Polym. 344, 122521 (2024). [DOI] [PubMed] [Google Scholar]

[R28] 28.Maiti D., Saha A., Devi P. S., Surface modified multifunctional ZnFe₂O₄ nanoparticles for hydrophobic and hydrophilic anti-cancer drug molecule loading. Phys. Chem. Chem. Phys. 18, 1439–1450 (2016). [DOI] [PubMed] [Google Scholar]

[R29] 29.Zhou Y., Wang R., Chen B., Sun D., Hu Y., Xu P., Daunorubicin and gambogic acid coloaded cysteamine-CdTe quantum dots minimizing the multidrug resistance of lymphoma in vitro and in vivo. Int. J. Nanomedicine 11, 5429–5442 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R30] 30.Zhang W., Tung C. H., Real-time visualization of lysosome destruction using a photosensitive toluidine blue nanogel. Chem. Eur. J. 24, 2089–2093 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] 31.Xie H., Zeng F., Wu S., Ratiometric fluorescent biosensor for hyaluronidase with hyaluronan as both nanoparticle scaffold and substrate for enzymatic reaction. Biomacromolecules 15, 3383–3389 (2014). [DOI] [PubMed] [Google Scholar]

[R32] 32.Kotla N. G., Bonam S. R., Rasala S., Wankar J., Bohara R. A., Bayry J., Rochev Y., Pandit A., Recent advances and prospects of hyaluronan as a multifunctional therapeutic system. J. Control. Release 336, 598–620 (2021). [DOI] [PubMed] [Google Scholar]

[R33] 33.Zhang W., Zhang Z., Tung C.-H., Beyond chemotherapeutics: Cisplatin as a temporary buckle to fabricate drug-loaded nanogels. Chem. Commun. 53, 779–782 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[R34] 34.Wallat J. D., Harrison J. K., Pokorski J. K., pH responsive doxorubicin delivery by fluorous polymers for cancer treatment. Mol. Pharm. 15, 2954–2962 (2018). [DOI] [PubMed] [Google Scholar]

[R35] 35.Wang J., Bhattacharyya J., Mastria E., Chilkoti A., A quantitative study of the intracellular fate of pH-responsive doxorubicin-polypeptide nanoparticles. J. Control. Release 260, 100–110 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

A dual chemodrug-loaded hyaluronan nanogel for differentiation induction therapy of refractory AML via disrupting lysosomal homeostasis

Shilin Xu

Tao Wang

Xuechun Hu

Hong Deng

Yiyi Zhang

Lei Xu

Yang Zeng

Jia Yu

Weiqi Zhang

Lin Wang

Haiyan Xu

Roles

Abstract

INTRODUCTION

RESULTS AND DISCUSSION

Fig. 1. Preparation and characterization of HA/Cis/Dau.

Fig. 2. Cellular uptake and chemotoxicity of the nanogels in vitro.

Fig. 3. Evaluation of the HA/Cis/Dau nanogel in AE & CKITD816V mouse model.

Fig. 4. Safety evaluation of HA/Cis/Dau nanogels in vivo.

Fig. 5. The differentiation-inducing effects of HA/Cis/Dau on Kasumi-1 cells.

Fig. 6. Metabolomic analysis of the AML cells after different treatments.

Fig. 7. Effects of HA/Cis/Dau in the BM-derived primary blasts from patients with AML with different AML subtypes.

Fig. 8. Schematic illustration of the differentiation induction effects of HA/Cis/Dau in refractory AML.

MATERIALS AND METHODS

Materials

Cell culture

Preparation and characterization of nanogels

Drug release experiment

Cell viability assay

Cellular uptake assay

Cell apoptosis assay

Cell differentiation assay in vitro

Cathepsin B assay

Galectin-3 staining

ROS detection

Proteome profiler array

Cell metabolism

NAD+/NADH assay

Treatment in AE & CKITD816V mice

Survival experiment

Pharmacodynamic experiment

Cell differentiation in vivo

Pharmacokinetic experiment in vivo

Toxicity evaluation of HA/Cis/Dau

Blood cell analysis

Histological and immunofluorescent staining

Patient samples

Statistical analysis

Acknowledgments

Supplementary Materials

This PDF file includes:

REFERENCES AND NOTES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Fig. 3. Evaluation of the HA/Cis/Dau nanogel in AE & CKIT^D816V mouse model.

NAD⁺/NADH assay

Treatment in AE & CKIT^D816V mice