*3.7. Calcium Phosphates Nanocarriers*

Due to its biocompatibility, biodegradability, pH responsive function, and can encapsulate in a variety of drugs in the matrix, calcium phosphate (CaP) was engineered as a drug delivery nanocarrier almost 50 years ago. The CaP nanocarriers for cancer imaging, therapy, and theranostics have been used for loading probes, nucleic acids, anticancer drugs, and photosensitizers. Moreover, they do not release the drug in the physiological plasma condition and release the drugs only in the acidic tumor environment. CaP NPs offer great biocompatibility in cancer therapy. CaP NPs loaded with caffeic acid, chlorogenic acid, or cisplatin were used in the presence of alginate polymer to minimize the burst release of the drugs. The drugs encapsulated in the CaP NPs exhibited anti-cancer activity in the concentration dependent manner [149]. CaP NPs are now being developed as non-viral transfection agents by adjusting the ratio of Ca and P molar ratio. Poly (L-Lysine) was used as a surface additive to optimize the transfection with plasmid DNA encoding a green fluorescent protein in the MC3T3 E cells (pre-osteoblastic). The nanosystem was less cytotoxic than the commercial viral carrier. OSA cells were four times more easily transfectable than pre-osteoblastic cells [150].

In a novel approach, bone substitute material, CaP was used as a scaffold for the resection of bone tumor. The CaPbeads were used for the delivery of cisplatin, doxorubicin and cis-diamminedichloroplatinum (CDDP). Doxorubicin was released continuously for 40 days whereas CDDP was burst released. The beads demonstrated cytotoxicity against MG-63 cells and proved promising for the therapy of OSA [151]. Functionalization of CaP with bioactive agents is a promising strategy in the bone targeted OSA therapy. The R enantiomer of 9-hydroxystearic acid (9R-9-HAS) inhibits tumor proliferation. Hence, 9R-9-HAS was incorporated in the CaP nanocrystals that modulated the cytotoxic effects on the OSA cells. The proliferation was reduced in the tumor cells by the increase of tumor necrotic factor [152]. Similarly, hydroxyapatite (a natural form of calcium apatite) NPs doped with selenium can fill the bone defects caused by tumors. The selenium released from the bone calcium-based structures induced the apoptosis of bone cancer cells by generating ROS. Additionally, the systemic toxicity was educed and tumor formation was inhibited [153]. In a similar, but novel approach, hydroxyapatite NPs were loaded with medronate, a bisphosphonate for targeting the bone cancer, and JQ1 as a small molecule bromodomain inhibitor as a chemotherapeutic. Medronate NPs had a high affinity for the hydroxyapatite. The NPs loaded with both JQ1 and medronate were cytotoxic against OSA cells in the 2-D culture and were completely compatible with the fibroblasts. OSA cells internalized the JQ1 loaded NPs efficiently [154].

#### *3.8. Other NPs*

Some other nano mediated drug delivery systems have been explored for the delivery of the drug in the treatment of OSA. Other therapeutic options involve the targeting of the surface expressed receptors on the OSA cells. CXCR1 marker is overexpressed on the

tumor cells and in OSA and is related to the chemotherapy resistance. CXCR1 targeting peptide was anchored to the magnetic NPs loaded with cisplatin. The NPs inhibited the cancer growth and prevented metastasis of the cancer cells to the pulmonary area [155]. Multi-functional micelles were developed and were loaded with curcumin because of its potential as an antitumor moiety. The micelles were synthesized by using amphiphilic alendronate-HA-octadecanoic acid. The nanomicelles were studied for their efficacy in OSA along with their bone affinity profile. Nanomicelles adhered to the bone because of the composition and released curcumin in a sustained manner. The cytotoxic effect of the nanostructures was pronounced [156]. Polymeric micelles are now being explored for photodynamic therapy by using potential photosensitizers for OSA. Zinc phthalocyanine is a dynamic photosensitizer with excellent photochemical properties. The poor solubility of zinc phthalocyanine was rectified by incorporating it in the poly(ethylene glycol)-pol(2- (methylacryloyl)ethylnicotinate)(PEG-PMAN) coblock micelles. ROS was significantly increased after light irradiation and exhibited 100% cytotoxicity as compared to the free photosensitizer [157].

Similarly, doxorubicin loaded self-assembled micelles were developed from RGD block copolymer poly(ethylene glycol)-block-poly (trimethylene carbonate). The half maximal inhibitory concentration was low as compared to the non RGD nanostructure that highlighted that RGD NPs have high cell targeting ability and anti-tumor effect in OSA [158]. In another similar approach, doxorubicin was loaded in the acid sensitive micelles for the OSA therapy. Hydrophilic D-aspartic acid octapeptide is a very promising micelle corona. Polymeric micelle was stabilized and loaded with the drug by an acid sensitive hydrazine bond. The stability of the polymeric micelles was increased by the increase in the concentration of aminoundecanoic acid to regulate the hydrophilic and hydrophobic ratio. Furthermore, the cytotoxicity was enhanced for the Saos-cells [159]. The polymeric micelles are also being investigated for the anti-cancer drug PENAO (4-(*N*-(*S*-penicillaminylacetyl) amino) which is currently in clinical trials for solid tumors. Direct PENAO polymeric micelles were developed by amidation reaction followed by polymerization with poly(ethylene glycol methyl ether methacrylate) as comonomer and poly(methyl methacrylate) (pMMA) as chain transfer agent, resulting in a coblock polymer. PENAO was readily available to actively target the mitochondria and inhibit cancer. Hence, it can provide a rationale platform for the OSA treatment [160].

OSA cells overexpress HER-2 receptors, thus making HER-2 a target for anti-HER-2 antibody trantuzumab. A nanomaterial structure of graphene oxide (GO) was developed and anchored with anti-HER-2 antibody by covalent bonding. The graphene nanostructure induced cell apoptosis by oxidative stress and leas to the formation of necroptosome. It also elevated the survival rate in animals, thus providing a promising curative therapy for OSA [161]. Alternatively, chitosan NPs were functionalized with GO for delivering siRNA to the OSA Saos-2 and M63 cells. ROS assay demonstrated the biocompatibility of nanoconjugate system and released siRNA in a controlled manner to the tumor site. Expression of inflammatory cytokines was reduced and the cancer cells were killed followed by the uptake in the cells [162].

Another nanosystem is dendrimer, which was reported to inhibit OSA. Dendrimer comprised of amphiphilic block copolymer poly (ethylene glycol)-poly (2-(methylacryloyl) ethylnicotinate)(PEG-PMAN) was synthesized and loaded with zinc phthalocyanine, used as a photosensitizer. The dendrimers elevated the ROS levels upon irradiation with light and killed OSA cells with high effectiveness [157]. Likewise, graphene-based dendrimers were developed to carry magnetic moiety for the delivery of multiple drugs in OSA. DOX and melatonin were coloaded in the branched nanostructures. Studies on Saos-2 and MG-63 osteosarcoma cells exhibited the down regulation of anti-apoptotic components and hence increased cytotoxicity [163]. The PAMAM dendrimers were mounted on the multiwalled carbon nanotubes and explored for the cytotoxicity to the OSA MG63. The nanoconjugate system was stable and biocompatible. The system also decreased the cellular

toxicity by 70% which was previously very high for the multiwalled carbon nanotubes (MWCNTs) [164].

Currently, exosomes derived from mesenchymal stem cells are gaining interest in the treatment of OSA. In one such study, DOX was loaded in the exosomes and was analyzed for the in vitro uptake in the MG-63 cells. Exosomes exhibited high infiltration in the MG-63 cells but low uptake in the myocardial H9C2 cells, hence proving to be promising for OSA targeted delivery [165]. Figure 4 highlights some of the nanostructures designed for the drug and gene delivery to OSA.

Recently, new trends are being explored for the treatment of various pathologies including OSA, based on self-assembling peptides. Such peptides can be explored by adjusting their peptide sequence, hence providing an opportunity for the generation of peptide of desired characters. Self-assembly of peptides creates a complex structure of high order for exploration in nanobiotechnological applications [166]. Currently, peptide nanofibrils are gaining interest as they disassemble inside the body and alter or support tissue growth, to make them free of any foreign material [167]. Similarly, ultra-short peptide hydrogels have been found to be efficient in delivering the drug to the cancerous cell. Such peptides perform a dual functions; initiate the growth of new cells and kill the cancerous cells [168].

It is now being studied that changing the peptide molecular properties might affect its interaction with small drugs and influence the release of the drug. The peptides being explored also undergo cytocompatibility studies to affirm their use as a drug delivery tool for biomedical use. The peptides have been found cytocompatible and do not illicit immune response. However, this novel idea still needs extensive research in the field of oncology. The future holds various horizons to be explored for the treatment of OSA.

#### **4. Conclusions, Challenges, and Perspectives**

Over the past few years, a significant number of targeted nanomaterials have been established for the diagnosis and treatment of malignant bone tumors such as OSA. It is imperative to provide a better understanding of the fundamental concepts involved in the design and application of nanoparticles for diagnosis, treatment, or the combination of imaging and therapeutics in various clinical circumstances, following this remarkable progress in the advancement of nanotherapeutic and imaging methods for cancer detection and treatment. OSA has rapidly metastasizing ability and proves challenging for the treatment rationales. Nanotherapeutics being developed for the OSA include metallic, lipid, polymeric, magnetic and stimuli-sensitive drug delivery systems. Nanotherapeutics improve the safety and compatibility profile in the diseases by minimizing the off-target accumulation. However, tumor biology itself plays a critical role and needs to be studied extensively for the outcomes in the case of nanoparticles therapy. The majority of the nanostructured approaches are in the cellular stages of drug delivery and need to be translated into clinical trials after extensive research. While these targeted NPs showed satisfactory benefits in OSA diagnosis and therapy, there are still difficult problems to solve in the future. For instance, in vivo verification of nanoparticles, and especially subsequent toxic evaluation and bone tissue targeted delivery for either cancer bone metastasis or other bone diseases still require further and extensive experiments to accelerate their potential clinical implementation. Some nano polymeric materials are not very strongly cytotoxic and it can also be expected that they will be offered to humans in the coming years. Nanotechnology is expected to play a pivotal role in future OSA diagnostics. With the development in technology, more powerful diagnostic techniques such as multimodal imaging can be seen in the coming days. Physicists, chemists, engineers, biologists, and clinicians, motivated by the rapid and encouraging developments in nanotechnology, will continue to challenge themselves to design innovative and efficient nanosystems for cancer diagnosis and treatment.

**Author Contributions:** The manuscript was written with contributions from all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Research Institute of Industrial Science & Technology (RIST), Republic of Korea, for which the authors are very grateful.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**


#### **References**

