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Antitumor Effect by Hydroxyapatite Nanospheres: Activation of Mitochondria-Dependent Apoptosis and Negative-Regulation of Phosphatidylinositol3-Kinase/Protein Kinase B Pathway Huan Zhao, Chengheng Wu, Dong Gao, Suping Chen, Yuda Zhu, Jing Sun, Hongrong Luo, Kui Yu, Hongsong Fan, and Xingdong Zhang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01996 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018
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Antitumor Effect by Hydroxyapatite Nanospheres: Activation of Mitochondria-Dependent Apoptosis and Negative-Regulation of Phosphatidylinositol3-Kinase/Protein Kinase B Pathway
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Huan Zhao, Chengheng Wu, Dong Gao, Suping Chen, Yuda Zhu, Jing Sun, Hongrong Luo, Kui Yu,
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†, ‡
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Hongsong Fan,* Xingdong Zhang
†
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, 610064, P.
R. China. ‡
Institute of Atomic and Molecular Physics, Sichuan University, Sichuan 610065, China
*E-mail:
[email protected] ACS Paragon Plus Environment
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ABSTRACT: Hydroxyapatite nanoparticles (HA NPs) have been acknowledged for their benign biocompatibility and proliferation inhibition effect on tumor cells, attracting considerable attention for tumor therapeutics without late effects. However, unnoticeable tumor cytotoxicity of HA NPs limited the final clinical therapeutic efficacy. Herein, a two-phase synthetic approach was developed to synthesize sphere-like HA NPs by varying the conventional growth habit of HA precipitate. We present our in vitro and in vivo experimental evidence that the spherical HA NPs have surprisingly-high inhibitory activities against tumor cells. We demonstrate further, based on our experimental data, that the underlying cause for the death of the tumor cells is related to two concurrent pathways, mitochondria-dependent
apoptosis
pathway
and
negative-regulation
of
phosphatidylinositol3-kinase/protein kinase B (PIK3/AKT) pathway. The present study indicated that HA nanospheres can be engineered as non-toxic specific inhibitors for efficient tumor therapeutics with nano-biomaterials.
KEYWORDS: hydroxyapatite, nanomaterials, tumor therapy, cell apoptosis, biomaterials
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For patients with tumor, traditional chemotherapy is the principal method to control tumor growth and inhibit metastasis. However, the nature of chemotherapy means that while damaging tumors it also damages certain normal tissues. So those patients frequently tolerated excruciating side effects, such as nausea/vomiting,
1
alopecia,
2
renal dysfunction,
3
myelosuppression,
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cardiomyopathy.
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Therefore, efficient and noninvasive therapy has attracted tremendous research attention aiming at relieving the sufferings of the patients. As expected, the biomedical and engineering world has present great increase in the use of nanoparticles (NPs) for nontoxic anti-tumor activities. Numerous clinical evidence has proven that assorted NPs assisting in killing the tumor cells and preventing tumor recurrence.
6-8
With the prevalence of NPs being incorporated in off-the-shelf anti-tumor
products, our exposure to NPs will correspondingly increase. 9
Hydroxyapatite (HA) is the principal inorganic constituent of mammalian bones, which has been used extensively and successfully as tissue repair and regenerative materials multifunctional medicine 12-14
effects.
11
l0
as well as
because of its excellent biocompatibility and outstanding biological
It is well known that the form of HA crystals frequently encountered is hexagonal, with cell 15
parameters of a=b=9.432 Å, c=6.881 Å, γ=120 °. 16-18
associated with the macro or micro structure.
And the main biological effects of HA were
Notably, when particle diameter is less than 100 nm,
HA has significant prohibitory effects on the metabolic viability of tumor cells instead of conventional biocompatibility.
19-21
These findings highlight the tremendous effect played by the HA NPs in the
cellular response, which could not only efficiently massacre tumor cells, but also exhibit no toxicity in certain normal tissues.
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This makes HA NPs a highly promising choice for tumor therapy so as to
avoid torturous late effects generated by traditional chemotherapy drugs. Although plentiful reports
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19, 23-25
confirmed the antitumor function of HA NPs, majority experiments settled for cell level in vitro,
the exact mechanism involved in tumor suppression by HA NPs remained vagueness. More importantly, few systematic experiments verify the inhibition effect of solid tumor, and the antitumor efficiency didn’t reach a high level in reality comparing with conventional antitumor drugs. Therefore, enhancement of antitumor activity of HA NPs is immensely meaningful to its further biological utilizations. In previous reports, researches demonstrated the antitumor effect of HA NPs might be related to the
intrinsic
functionality.
physicochemical characteristics,
26-29
including
crystal
size,
morphology,
surface
Many studies also attempt to change the structure of HA NPs in order to improve the
inferior proliferation inhibition effect on tumor cells, but the improvement was very limited. Due to initial preferential orientation of HA crystal,
30, 31
it was easier to obtain needle-like or rod-like HA 32-35
nanocrystals with imbalance aspect ratio in the process of growth.
The undiversified structure
exerted poor adjustability as well as showed imperfect performance. Certainly, a few reports are also available on spherical HA NPs, however, the as-obtained structures are reported to have diameters up to several hundreds of nanometers with irregular assembled morphologies,
36-39
which cannot be
defined authentic sphere-like nanostructure. Thus, the development of high-quality spherical HA NPs while overcoming the above limitations would be highly desirable. The inventive sphere-like HA NPs might produce unusual biological effects, specifically outstanding anti-tumor activities. In our study, we devised a simple and efficient system and obtained real sphere-like HA nanoparticles. On account of altering habit of HA by reconstruction of atoms and recrystallization of amorphous precursor in the two-phase system, the as-synthesized HA NPs are with well-defined
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spherical structure and sub-50 nm size. As we expected, the experimental results demonstrated the new HA nanospheres exhibited highly efficient antitumor properties both in vitro on the 4T1 cell line, and in vivo on the 4T1 xenograft model. Compared with the traditional chemotherapeutics, HA nanospheres not only has highly compatibility to normal tissues, but also has excellent antitumor activities, therefore, demonstrate great potential in clinical application. In addition, to gain an understanding of the mechanism of antitumor, we systematically explored the in vitro cellular uptake, cytotoxicity and apoptosis-induced phenomenon, apoptotic molecular signaling pathway and the in vivo tumor growth inhibition. Furthermore, these results showed that mitochondria-dependent apoptosis pathway and negative regulation of PIK3/AKT pathway might be crucial mechanisms contributing to the enhancement of apoptosis of tumor cells. This study may offer a preeminent understanding of the scientific molecular mechanism of HA NPs-induced apoptosis as well as fill the blank about signaling pathway activation on the feedback of HA NPs. Results and discussion Synthesis and characterization of HA nanospheres. Figure 1A illustrates a possible formation mechanism of the HA nanospheres. In traditional homogeneous system, vigorous agitation introduced supersaturation of partial reactant ions, sequentially generated small precursors. Due to initial oriented growth, these precursors grew along specific c axis and finally evolved rod-like HA 40
NPs. In our designed two-phase (toluene/water) system, vigorous stirring would generate interfacial 41
spontaneous emulsification, thus burst into numerous crystal nucleus.
When the agitation was
interrupted, emulsion droplets disappeared and crystal nucleus assembled into precursors.
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Stable
interface was conductive to form precursor with well-define sphere-like structure. To the contrary,
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Figure 1. (A) Schematic illustration of the synthesis of HA NPs. (B) Low-magnification TEM image, inset: corresponding HRTEM image. (C) XRD patterns. (D) FTIR spectra. (E) EDS patterns of HA NPs.
high temperature leaded to dramatic thermodynamic fluctuations in the half-homogeneous interface 43
system,
the assembly process of the precursors will be disrupted so that we cannot obtain inerratic
sphere-like precursor. Next, as-grown precursor and residuary reactant diffused to the interface, crystallization process continued by atom reconstruction. Since emulsion-assisted nucleation consumed most reactants, deficient feedstock was participated in growth stage. Thus, spherical morphology of precursors was duplicated, and HA nanospheres were finally formed. The detailed experimental procedures are given in the experimental section. TEM images as compelling evidence suggest the above mechanism for the synthesis of HA NPs (Figure 1B). Different from the previous reports and the as-synthesized products in homogeneous and half- homogeneous system, we obtained sphere-like HA nanoparticles in a two-phase approach by controlling nucleation and crystal growth processes. The final size of the HA nanospheres were only 10-30 nm. Afterwards, the X-ray diffraction (XRD) in Figure 1C indicates all the diffraction peaks can be indexed to a phase-pure Ca10(PO4)6(OH)2 (JCPDS 09-0432) with a hexagonal structure. Importantly, the relative intensity of (002) peak was significant decline in the two-phase system, indicating that the preferential growth may be restricted during the stage of recrystallization.
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The change of growth habit was helpful to
fabricate perfect sphere-like structure instead of traditional rod-like or needle-like HA NPs. Afterwards, Figure 1D shows the appearing functional groups of HA. It is worth noting that -COO groups can be
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Figure 2. Specific antiproliferative activity. Four kinds of tumor cells exposed to HA nanospheres at indicated concentrations (30, 90 and 150 µg/mL). (A) Cell viability detected by MTT. (B) Cellular proliferation inhibition rate on 5 d, (*p
