Specific cancer stem cell-therapy by albumin nanoparticles functionalized with CD44-mediated targeting

Abstract

Background: Cancer stem cells (CSCs) are highly proliferative and tumorigenic, which contributes to chemotherapy resistance and tumor occurrence. CSCs specific therapy may achieve excellent therapeutic effects, especially to the drug-resistant tumors.

Results: In this study, we developed a kind of targeting nanoparticle system based on cationic albumin functionalized with hyaluronic acid (HA) to target the CD44 overexpressed CSCs. All-trans-retinoic acid (ATRA) was encapsulated in the nanoparticles with ultrahigh encapsulation efficiency (EE%) of 93% and loading content of 8.37%. TEM analysis showed the nanoparticles were spherical, uniform-sized and surrounded by a coating layer consists of HA. Four weeks of continuously measurements of size, PDI and EE% revealed the high stability of nanoparticles. Thanks to HA conjugation on the surface, the resultant nanoparticles (HA-eNPs) demonstrated high affinity and specific binding to CD44-enriched B16F10 cells. In vivo imaging revealed that HA-eNPs can targeted accumulate in tumor-bearing lung of mouse. The cytotoxicity tests illustrated that ATRA-laden HA-eNPs possessed better killing ability to B16F10 cells than free drug or normal nanoparticles in the same dose, indicating its good targeting property. Moreover, HA-eNPs/ATRA treatment decreased side population of B16F10 cells significantly in vitro. Finally, tumor growth was significantly inhibited by HA-eNPs/ATRA in lung metastasis tumor mice.

Conclusions: These results demonstrate that the HA functionalized albumin nanoparticles is an efficient system for targeted delivery of antitumor drugs to eliminate the CSCs.

Keywords: All-trans-retinoic acid; CD44; Cancer stem cells; Cationic albumin; Hyaluronic acid.

Fig. 1

In vitro characteristics of NP formulations. a, b Diameter and zeta potential measurements of NP formulations. c Different nanoparticles visualized by transmission electron microscopy (TEM), scale bar: 50 nm (arrows indicate the coated HA layer). d The encapsulation efficiency and drug loading of NP formulations. Results are shown as the means ± SD (n = 3). e Release profiles of ATRA from NP formulations in PBS with 10% (v/v) ethanol. Results are shown as the means ± SD (n = 3). f Differential scanning calorimetry (DSC) thermograms of ATRA formulations

Fig. 2

Stabilities evaluation of ATRA loaded HA-eNPs. Continually determinations of a the diameter, b the zeta potential, c the encapsulation efficiency, and d HA density of HA-eNPs within 4 weeks, respectively. Results are shown as the means ± SD (n = 3)

Fig. 3

Fluorescence analysis of uptake of HA-eNPs/ATRA in B16F10 cells. a Fluorescence images of uptake of coumarin-6-labeled NPs at different ATRA concentrations (4 h). b Fluorescence images of uptake of coumarin-6-labeled NPs at different time points (5 μM ATRA). Fluorescence signals were observed by fluorescence microscopy. Scale bar represents 50 μm

Fig. 4

In vitro evaluation of detecting the targeting of HA-eNPs to CD44. a, b Coumarin-6 loaded HA-eNPs were incubated with CD44-enriched B16F10 cells or CD44-low expressing MCF-7 cells, respectively. Fluorescence signals were observed by confocal microscopy. c Flow cytometry analysis showing the cellular uptake percentage of the B16F10 and MCF-7 cells after incubation with free coumarin-6, coumarin-6 loaded NPs, or HA-eNPs. Data are shown as the means ± SD, ^**P < 0.01, ns not significant (n = 3). d, e B16F10 cells were preincubated with anti-CD44 antibody and followed by incubating with HA-eNPs for 4 h. Fluorescence signals were observed by confocal microscopy and flow cytometry. Data are shown as the means ± SD, ^**P < 0.01 (n = 3)

Fig. 5

In vitro antitumor effects of NP formulations on the B16F10 cells. a Morphological changes of B16F10 cells after treatment with various ATRA formulations. Scale bar represents 50 μm. b MTT viability assay for B16F10 cells after treatment with various ATRA formulations. Results are shown as the means ± SD (n = 3). c Apoptosis of B16F10 cells induced by various ATRA formulations (5 μM) (arrows indicate cell apoptosis). Scale bar represents 20 μm. d Flow cytometry analysis of B16F10 cell apoptosis induced by ATRA formulations. Results are shown as the means ± SD (n = 3)

Fig. 6

Fluorescence imaging of ATRA-loaded NP formulations in mice. a Time-dependent intensity images of fluorescence distribution in mice (arrows indicate accumulation of HA-eNPs in tumor-bearing lungs). b Fluorescence images of major organs at 8 h post-injection of NP formulations in mice. c Fluorescence microscopy images of tumor-bearing lung tissue section. Scale bar represents 50 μm

Fig. 7

HA-eNPs/ATRA decreased the tumorigenicity of CD44-enriched B16F10 cells. a Side population (SP) changes in B16F10 cells after treatment with various ATRA formulations. b A diagram of the tumorigenicity assessment. In the experiment, four flanks of each mouse were injected with the same treated or untreated B16F10 cells. c Representative photo of tumors 24 days after subcutaneous implantations of ATRA formulations treated or untreated B16F10 cells (arrows indicate tumor nodules). d, e Tumor occurrences and weights measured 24 day after implantation. Results are shown as the means ± SD (n = 16)

Fig. 8

HA-eNPs/ATRA enhanced the inhibition of tumor growth in the in situ lung metastasis tumor-bearing mice. a Images of the B16F10-bearing lungs on day 24 after five consecutive i.v. injections of NP formulations (n = 5). b Antitumor effects of various treatments evaluated according to B16F10-bearing lung weight (n = 5). c Histological staining of the B16F10-bearing lungs after various treatments (arrows indicate tumor nodules, n = 5)

Fig. 9

Safety evaluation of ATRA-loaded NP formulations. Microscopic images of hematoxylin and eosin (H&E) staining of organs (liver, spleen, lung, kidney, heart and brain) from mice treated with ATRA-loaded NP formulations. No abnormality was observed. Scale bar represents 50 μm