Transferrin Modified GSH Sensitive Hyaluronic Acid Derivative Micelle to Deliver HSP90 Inhibitors to Enhance the Therapeutic Efficacy of Brain Cancers

Abstract

Herein, GSH-sensitive hyaluronic acid-poly(lactic-co-glycolic acid) (HA-SS-PLGA) was synthesized. Surface modification of PLGA with hyaluronic acid produced a highly stable micelle at physiological pH while a micelle was destabilized at a higher GSH level. Fluorescence microscopy results showed that rhodamine-encapsulated micelle was taken up by brain cancer cells, while competitive inhibition was observed in the presence of free HA and free transferrin. In vitro cytotoxicity results revealed that transferrin-targeted nanoformulated AUY922 (TF-NP-AUY922) shows higher cytotoxicity than either free AUY922 or non-targeted AUY922-loaded micelles (NP-AUY922). In comparison to the control groups, free AUY922, TF-NP-AUY922 or NP-AUY922 treatment revealed the upregulation of HSP70, while the expression of HSP90 client proteins was simultaneously depleted. In addition, the treatment group induced caspase-dependent PARP cleavage and the upregulation of p53 expression, which plays a key role in apoptosis of brain cancer cells. In vivo and ex vivo biodistribution studies showed that cypate-loaded micelle was taken up and accumulated in the tumor regions. Furthermore, in vivo therapeutic efficacy studies revealed that the AUY922-loaded micelle significantly suppressed tumor growth in comparison to the free AUY922, or control groups using tumor-bearing NOD-SCID mice. Moreover, biochemical index and histological analysis revealed synthesized micelle does not show any significant cytotoxicity to the selected major organs. Overall, a synthesized micelle is the best carrier for AUY922 to enhance the therapeutic efficiency of brain cancer.

Keywords: GSH-sensitive micelle; brain cancer; hyaluronic acid; poly(lactic-co-glycolic) acid.

Scheme 1

(A) Scheme of HA-SS-PLGA synthesis and (B) transferrin and CD44 receptor-mediated cellular uptake of AUY922-loaded micelle.

Figure 1

¹H-NMR of HA-SS-PLGA and PLGA-Cys in DMSO-d6/D2O and DMSO-d6, respectively.

Figure 2

FTIR spectrum of HA-SS-PLGA and its precursors.

Figure 3

(A) Particle size using DLS, (B) TEM image of empty micelle and AUY922-loaded micelle, (C) Particle size stability in DI water and 10% FBS, (D) in vitro drug release in the presence and absence of GSH and (E) TEM image of AUY922-loaded micelle in the presence of GSH (5 mM).

Figure 4

MTT in vitro cytotoxicity results using P5 and P5-TMZ-R cancer cells (A) HA-SS-PLGA and PLGA-Cys; (B) and (C) free AUY922, TF-NP-AUY922, and NP-AUY922, (D) free AUY922, TF-NP-AUY922 and NP-AUY922 using U87 brain cancer cells, (E–G) representative images of the crystal violet-stained colonies using P5, P5-TMZ-R, and U87 brain cancer cells, respectively. (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5

(A) Apoptosis assay of free AUY922, NP-AUY922, and TF-NP-AUY922 after 72 h of incubation at a concentration of 100 nM for free or nanoformulated AUY922. (B) Expression of P53, activation of caspase 3 (cleaved caspase 3), activation of PARP (cPARP) and β-actin proteins in P5, P5-TMZ-R, and U87 brain cancer cells after treating for 48 h using 100 nM free AUY922, NP-AUY922, and TF-NP-AUY922. (C) Cell cycle analysis of free AUY922, NP-AUY922, and TF-NP-AUY922 using P5, P5-TMZ-R, and U87 brain cancer cells after 48 h of incubation at a concentration of 100 nM for free or nanoformulated AUY922. In (B), the actin in the P5/P5-TMZ-R part of the experiment is identical as shown in Figure 7A, and the actin in the U87 part of the experiment is identical as shown in Figure 7A,B, because they are part of the same original blots probed with different antibodies of interest. The figures are re-organized as presented to better align with the data demonstrated in the Results section.

Figure 5

(A) Apoptosis assay of free AUY922, NP-AUY922, and TF-NP-AUY922 after 72 h of incubation at a concentration of 100 nM for free or nanoformulated AUY922. (B) Expression of P53, activation of caspase 3 (cleaved caspase 3), activation of PARP (cPARP) and β-actin proteins in P5, P5-TMZ-R, and U87 brain cancer cells after treating for 48 h using 100 nM free AUY922, NP-AUY922, and TF-NP-AUY922. (C) Cell cycle analysis of free AUY922, NP-AUY922, and TF-NP-AUY922 using P5, P5-TMZ-R, and U87 brain cancer cells after 48 h of incubation at a concentration of 100 nM for free or nanoformulated AUY922. In (B), the actin in the P5/P5-TMZ-R part of the experiment is identical as shown in Figure 7A, and the actin in the U87 part of the experiment is identical as shown in Figure 7A,B, because they are part of the same original blots probed with different antibodies of interest. The figures are re-organized as presented to better align with the data demonstrated in the Results section.

Figure 6

Cellular uptake of rhodamine B-encapsulated micelle and competitive inhibition assay using free transferrin, hyaluronic acid, or both using (A) P5 and (B) P5-TMZ-R cancer cells. Note: free transferrin and hyaluronic acid were incubated with the P5-and P5-TMZ-R brain cancer cells for the 1 h before treating with the rhodamine B-encapsulated micelle. The scale bar is 100 μm.

Figure 7

(A) Expression levels of HSP90, HSP70, and β-actin proteins (B) expression of AKT, pAKT, ERK2, and β-actin proteins in P5, P5-TMZ-R, and U87 brain cancer cells after treatment for 48 h using 100 nM free AUY922, NP-AUY922, and TF-NP-AUY922. In (A), the actin in the P5/P5-TMZ-R part of the experiment is identical as shown in Figure 5B, and the actin in the U87 part of the experiment in both (A,B) are the same, which was identical as shown in Figure 5B, because they are part of the same original blots probed with different antibodies of interest. The figures are re-organized as presented to better align with the data demonstrated in the Results section.

Figure 8

Ex vivo fluorescence image of selected major organs for (A) NP-cypate and (B) TF-NP-cypate (at cypate dosage: 1 mg/kg of mice). Note: brain (B), heart (H), lung (Lu), liver (Li), kidney (K), pancreas (P), spleen (S), and tumor (T).

Figure 9

In vivo therapeutic efficacy of PBS (as a control), free AUY922, NP-AUY922, and TF-NP-AUY922 using U87 brain cancer-bearing NOD-SCID mice. The drugs were given on days 0, 3, 7, 10, and 13 (dosage: 20 mg/kg of mice for nanoformulated AUY922 and 40 mg/kg of mice for free AUY922). (A) Relative tumor volume, (B) selected images of the tumors collected on day 14, and (C) relative bodyweight of NOD-SCID mice. Data are presented as mean ± SD (n = 5). (** p < 0.01, *** p < 0.001). A one-tailed t-test was used for the p-value calculation for each control vs. treatment group.

Figure 10

Toxicity evaluation of free AUY922, NP-AUY922, and TF-NP-AUY922 in comparison to control groups after 14 days of injections (A) Liver function test, (B) Kidney function test, (C) H&E staining. Scale bar represents 200 μm and magnification 20×.