Nanomedicine in osteosarcoma therapy: Micelleplexes for delivery of nucleic acids and drugs toward osteosarcoma-targeted therapies
Journal Pre-proofs Review article Nanomedicine in osteosarcoma therapy: micelleplexes for delivery of nucleic acids and drugs for osteosarcoma-targete...
Journal Pre-proofs Review article Nanomedicine in osteosarcoma therapy: micelleplexes for delivery of nucleic acids and drugs for osteosarcoma-targeted therapy Miguel Pereira-Silva, Carmen Alvarez-Lorenzo, Angel Concheiro, Ana Cláudia Santos, Francisco Veiga, Ana Figueiras PII: DOI: Reference:
European Journal of Pharmaceutics and Biopharmaceutics
Received Date: Revised Date: Accepted Date:
16 July 2019 9 September 2019 31 October 2019
Please cite this article as: M. Pereira-Silva, C. Alvarez-Lorenzo, A. Concheiro, A. Cláudia Santos, F. Veiga, A. Figueiras, Nanomedicine in osteosarcoma therapy: micelleplexes for delivery of nucleic acids and drugs for osteosarcoma-targeted therapy, European Journal of Pharmaceutics and Biopharmaceutics (2020), doi: https:// doi.org/10.1016/j.ejpb.2019.10.013
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nanomedicine in osteosarcoma therapy: micelleplexes for delivery of nucleic acids and drugs for osteosarcoma-targeted therapy
Miguel Pereira-Silva1, Carmen Alvarez-Lorenzo3, Angel Concheiro3, Ana Cláudia Santos1,2 Francisco Veiga1,2, Ana Figueiras1,2*
1Department
of Pharmaceutical Technology, Faculty of Pharmacy, University of
Coimbra, Portugal;
2REQUIMTE/LAQV,
Group of Pharmaceutical Technology, Faculty of Pharmacy,
University of Coimbra, Coimbra, Portugal;
3Departamento
de Farmacología, Farmacia y Tecnología Farmacéutica, I+D Farma
(GI-1645), Facultad de Farmacia and Health Research Institute of Santiago de Compostela (IDIS), Universidade de Santiago de Compostela, Spain.
*Corresponding author: Dr. Ana Figueiras Phone: (+351) 239 488 431 Fax. (+351) 239 488 503 E-mail address: [email protected] Postal address: Faculty of Pharmacy, University of Coimbra, Azinhaga de Santa Comba, 3000-548, Coimbra, Portugal
Abstract Osteosarcoma(OS) represents the main cancer affecting bone tissue, and one of the most frequent in children. In this review we discuss the major pathological hallmarks of this pathology, its current therapeutics, new active biomolecules, as well as the nanotechnology outbreak applied to the development of innovative strategies for selective OS targeting. Small RNA molecules play a role as key-regulator molecules capable of orchestrate different responses in what concern cancer proliferation, migration and invasiveness. Frequently associated with lung metastasis, new strategies are urgent to upgrade the therapeutic outcomes and the life-expectancy prospects. Hence, the prominent rise of micelleplexes as multifaceted and efficient structures for small nucleic acid delivery and selective drug targeting is revisited here with special emphasis in ligandmediated active targeting. Future landmarks towards the development of novel nanostrategies for both OS diagnosis and OS therapy improvements are also discussed. Keywords Cancer targeting; osteosarcoma; doxorubicin; micelleplexes; gene delivery; active targeting Funding This work was supported by the grant FCT PTDC/CTM-BIO/1518/2014 from the Portuguese Foundation for Science and Technology (FCT) and the European Community Fund (FEDER) through the COMPETE2020 program.This work received financial support from National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência) through project UID/QUI/50006/2013, co-financed by European Union (FEDER under the Partnership Agreement PT2020). It was also supported by the grant FCT PTDC/BTM-MAT/30255/2017 (POCI-010145-FEDER-030255) from the Portuguese Foundation for Science and Technology (FCT) and the European Community Fund (FEDER) through the COMPETE2020 program. This research was also funded by MINECO (SAF2017-83118-R), Agencia Estatal de Investigación (AEI) Spain, Xunta de Galicia (Grupo de Referencia Competitiva ED431C 2016/008), and FEDER (Spain).
1. Introduction Cancer comprises one of the biggest challenges in nowadays’ Medicine, with an expected increase in new cases in the near future and strong impact at all levels of socio-sanitary care[1, 2]. Cancer burden in 2018 was estimated in its incidence and mortality, corresponding to 18.1 million new cancer cases and 9.6 million deaths related to this disease[2]. Osteosarcoma(OS) represents the commonest primary malignance of the bone tissue, affecting children and adolescents at much higher rate than other segments of population. This malignant bone tumor is characterized by the production of immature osteoid extracellular matrix and has a mesenchymal origin[3]. Whilst continuous efforts regarding therapies to improve and ameliorate clinical outcomes, including chemotherapy and limb salvage surgery have leveraged the 5-year survival rate up to >65% in localized resectable tumors, in the case of advanced and metastasized osteosarcoma the rate markedly decreases to ca. 20%[4]. Moreover, akin to other types of cancer, there is a remarked tendency for invasion and early metastasis, primarily to the lungs, worsening the therapeutic outcomes and life expectancy among patients diagnosed with this rare cancer. Several pathways and molecules of different origins may contribute to the instalment and progression of this disease. Bearing in mind that the medical therapy for this disease has met little changes throughout the years since the 1970’s, new therapies are urgently needed. Innovative therapies able to improve OS therapy efficiency and capable of lower the toxicity and adverse effects related to the conventional chemotherapy bestow categorical relevance, as potential advances are under development regarding cancer therapy in OS[5]. Novel drug delivery systems have met intensive developments, and nanotechnology applied to biomedicine has emerged as a multi-advantageous approach, since it can provide remarkable improvements in drug bioavailability
(enhanced solubility and stability), biodistribution and controlled release by not only prolonging drug systemic circulation, but also enabling the site specific delivery of the drug and its accumulation in tumor sites, thus achieving a selective targeting profile [6]. Polymer-based nanosystems are interesting platforms in different targeted therapies and the combination of both therapeutic and diagnostic molecules is being received with special enthusiasm as synergistic strategies for tackling tumor cells. RNA interference (RNAi) technology has provided interesting results in using small interfering molecules playing roles in cancer regulation processes. Henceforth, different nanocarriers have been explored as anti-cancer active platforms for OS eradication, and within the panorama of polymeric nanoparticles, micelleplexes arrived as interesting nanocarriers for gene delivery. Micelleplexes result from the interaction of genetic material with amphiphilic copolymers bearing one or more cationic blocks. The hydrophobic block confers an inner hydrophobic core ideal for hydrophobic drugs encapsulation, while the corona-forming hydrophilic block is usually made of polycations including polyethyleneimine (PEI), poly(2dimethylaminoethyl methacrylate) PDMAEMA and polyaminoacids, such as poly-Llysine (PLL) [7]. The high density of positive charges on the cationic block allows for efficient nucleic acid condensation. Charge neutralization strongly contributes to the selfassembly of the block copolymer leading to micelle-like nucleic acid-polymer nanocomplexes. The cationic properties ensure not only the establishment of electrostatic interactions with the negatively-charged phosphate groups of nucleic acids, hence mediating efficient nucleic acid complexation, but also interactions with the negativelycharged membranes, thus contributing to cellular uptake and endosomal escape. The buffer capacities of polycations allows for endosomal disruption and further cytosolic release of the nucleic acid payload[8, 9]. Besides, as multifunctional tunable
nanosystems, micelleplexes may be endowed with stimuli-responsive properties for nucleic acid release [10] and can be surface decorated with ligands for active targeting strategies and/or cell penetrating peptides for increased cell membrane penetration, maximizing nucleic acid delivery into cells[9]. 2. Osteosarcoma’s pathology and therapeutic regimen 2.1. Osteosarcoma’s pathological hallmarks The complexity of the pathological pathways associated to the initiation, proliferation and invasion of OS are undoubtedly still a challenge as for many other cancers. The interconnection among different underplayers and the wide number and scope of molecules, pathways and systems involved have been described to a certain extent in diverse literature. Wnt pathway is perhaps one of the most extensively studied in this specific type of cancer and, despite the need for further investigations, it appears to play an important role in what cancer cells growth is concerned. The canonical Wnt signalling is the most broadly studied and through its activation via a Wnt ligand, a signal cascade is triggered resulting in activation of specific mediators, followed by the dissociation of a proteic complex, thus resulting on the intracellular accumulation of the protein BCatenin, that further translocates to the nucleus stimulating the transcription of several genes, such as the c-myc and cyclin D1, enhancing cellular responses and potentially contributing to cell growth and proliferation[11]. It should be noted that cyclins are true key-players in the cell growth process and cell cycle regulation[12]. As a group of signal transduction pathways, there are other signalling mechanisms, also called the noncanonical Wnt signalling pathways, without B-catenin involvement, also important for several cellular mechanisms such as cytoskeleton and calcium levels regulation, but less studied. Notch signalling has also revealed an important pathway in which there is an
overexpression of Notch genes and ligands in OS cell lines with greater metastization capabilities, thus suggesting an important association between the tumour ability to metastasize and the important regulatory function of the notch pathway signalling [13]. Other significant pathways well-described in literature are the Ezrin pathway[14, 15] and the Ephrin-receptors mediated molecular signalling pathway[16, 17]. In fact, the probable role of ezrin, a member of the ERM (ezrin/radixin/moesin) group of proteins functioning as a membrane-cytoskeleton linker[18], in metastasis formation and cancer progression has been studied. Ezrin can activate NF-kappaB pathway leading to a EGF-induced epithelial-mesenchymal transition (EMT), instigating the tumor progression through a NF-kappaB-ezrin route[19]. Aberrant signalling is found in OS and receptor tyrosine kinases (RTKs) overexpression and implication in tumor progression is well documented, such as the Axl, EphB2 (ephrin B2 receptor), FGFR2 (fibroblast growth factor receptor 2), and Ret [20]. A growing body of evidence suggests that the extracellular matrix metalloproteinases (MMP), including MMP-1, MMP-2 and MMP-9, play a role in proliferation, invasion and metastasis of OS cells[21-23]. Mutations in the retinoblastoma (Rb) gene Rb-1 and p53 gene have long been identified as genetic alterations associated with the development of OS[24]. Interactions between the OS cells and the lung are of utmost importance for lung colonization and the establishment of metastasis, reinforcing the importance of the lung metastatic niche[5]. Despite the need for further investigations, some mediators have been identified, for instance IL-6 and CXCL8, the two possibly implied in tumor tropism[25]. An illustration of the principal pathways occurring in OS can be found in Figure 1. Besides, several potential prognosis biomarkers have been studied regarding their implications in the development of OS, metastasis formation and predictive of poor survival chances, such as apoptosis antigen 1 (Fas)-negative OS cells, which can evade normal elimination mechanisms and potentiate metastization [26, 27]
and nucleic acids, such as microRNA 183 downregulation implied in cancer migration and invasiveness, via Ezrin pathway[28]. Besides, vascular endothelial growth factor (VEGF) is a widely-touted prognostic marker, a key regulator of angiogenesis with significant clinical importance regarding OS, its progression and prognosis. Indeed, its overexpression in osteosarcoma patients appears to be an indicative of poor prognosis[29]. Therefore, a connection between the potential biomarker’s expression and either the increasing histopathological marks (increase of tumor size and/or metastatic processes) and clinical marks (advanced tumor grading and clinical staging and decreased overall survival) are not only relevant aspects regarding OS prognosis and evolution context, but also to the development of new targeted therapies based on such dysregulated pathological mechanisms, paving the way to new avenues for OS treatment, as discussed later in this paper.
< Figure 1 > Figure 1. Osteosarcoma view through cell proliferation, invasion and metastasis mechanisms including lung colonization and specific interaction with the tumoral niche.
2.2. Osteosarcoma therapy: from conventional to innovative therapies The entailed pathological features of OS appearance, progression and tissue-invasion abilities have undoubtedly painted OS as a highly malignant, prone-to metastasis, rapidlyspreading cancer, also related to relatively poor prognosis and clinical outcome. The overall exposure cycle from which the patient is affected by the disease, including risk factors, diagnosis, staging and preconized treatments have been widely outlined (Figure 2).
< Figure 2> Figure 2: Osteosarcoma: key facts and general perspective.
Despite the therapeutic arsenal, there is a significant percentage of unsuccessful cases, thus calling for new and more efficient strategies. The search for new molecular targets for therapeutic purposes has met significant disappointment and remains a significant challenge[30]. According to the American Cancer Society, the three main treatments applicable are surgery, chemotherapy and radiotherapy. Albeit surgical resection and combinational chemotherapy are incorporated in standard-care therapies, radiotherapy can be prescribed especially in the case of non-resectable tumors, in which tumor removal surgery cannot be done effectively after chemotherapy was given. Radiotherapy helps not only by contributing to the control of the expansion of cancerous cells, thus adding to the therapeutic success, but also to ameliorate and relieve the well-known symptoms associated with the cancer therapy itself[31]. Adjuvant chemotherapy has shown to improve the prognosis of OS patients and is usually administered via drug combos, such as dual administration of doxorubicin and cisplatin[4]. Significant progresses have been documented recently, regarding adjuvant chemotherapy in OS therapy, as other drug combinations have shown therapeutic advantageousness[32]. Whilst the general protocol of therapeutics in OS differ upon the type and evolution of the disease, the sequence of treatment is usually initiated by a period of before-surgery chemotherapy, which can be helpful in the case of non-resectable tumors, followed by the tumor-removal surgery and an additional adjuvant chemotherapy period. Nevertheless, the outcome for patients with advanced stages OS remains dismal, hence other strategies have been developed for paving the way to new treatment avenues[33]. Efforts to carry out new and innovative drug development strategies that leverage new research, and clinical trials have been
undertaken. Hence, different approaches with potential clinical relevance regarding OS therapy are under research focus. Immunotherapy has received considerable attention as an increasingly attractive strategy for OS treatment[5]. Nevertheless, clinical benefit has not yet shown to be sufficient, as therapeutic success is hampered by the rarity of this cancer, its heterogeneity and still insufficient knowledge regarding tumor specific-antigens as new targets[34]. Besides, the complexity of the immune system and immune-cancer cell biointeractions and resistance mechanisms developed by cancer cells have remained a challenge to overall improvements in immunotherapy response[35]. Special emphasis has been given to checkpoint inhibitors-based therapeutic approaches as well as to combined therapies to tackle the immunosuppressive microenvironment of OS[35, 36]. Cellular immunotherapy recurring to gene-engineered tumor-specific T-cells, such as T cell receptor (TCR)engineered T cells and chimeric antigen receptor T (CAR-T) cells [37] can upgrade functionality and precision of T cells[38] and, together with dendritic cell (DC) therapies [39], have shown increasing interest among the current innovative therapies under development. Another strategies including antibody-based ones such as monoclonal antibodies targeting insulin-like growth factor-1 receptor (IGF-1R)[40], insulin-like growth factor-2 receptor (IGF-2R)[41], anti-ganglioside GD2 antibody[42]
and
bispecific antibodies targeting IGF-1R and EGFR[43]have also been explored. Additionally, nucleic acids including ribonucleic acid interference (RNAi) molecules, such as micro RNAs (miRNAs) and small interfering RNAs (siRNAs) have been put into the bargain, as different studies have shown their potential roles mediating OS evolution[44]. However, their potential therapeutic action regarding degradation of messenger RNA (mRNA) through RNAi mechanism with the intent to regulate certain cellular pathways is hampered due to their physicochemical characteristics, such as large
molecular weight, poor lipophilicity, anionic properties due to negatively-charged phosphate groups, which put together along serum instability and increased susceptibility toward enzymatic degradation limits their bioavailability, cellular uptake, endosomal escape and the ultimate transfection efficiency. Therefore, advanced strategies for loading, transportation and delivery of nucleic acid-based therapies to cancer cells are demanded[45]. In this context, several strategies including modifications in nucleic acid chemistry (e.g. for stability enhancement)[46] and optimization of viral[47] and non-viral vectors[48] have been explored. Non-viral vectors draw several advantages such as decreased potential to trigger immune responses, easier manipulation and tunable properties, as opposed to viral ones[49]. Regarding OS targeting, several non-viral strategies are discussed below, with special emphasis on micelleplexes as suitable nanocarriers for nucleic acid delivery to cancer cells.
3. A big leap to nanomedicine: osteosarcoma-targeted therapies Nanomedicine has emerged as one of the standouts of biomedical research technology favouring diagnosis, therapy and management of several diseases. Drugs and other molecules can be successfully loaded into nanocarriers for targeted therapy[50]. For this purpose, several nanosystems – either viral and non-viral - have been developed in order to tackle cancer diseases and to improve the therapeutic outcomes. Nanotechnologybased cancer therapy strategies assume more and more a remarkable relevance in today’s health researches, much abiding to the necessity to enhance targeting efficiency, reduce both systemic adverse effects and drug dosage, among others. Henceforth, different nonviral strategies have been investigated in the context of cancer therapy, integrating different pharmacological and mechanistic approaches, and OS is not an exception. Both passive - taking advantage of enhanced permeability and retention (EPR) effect - and
active targeting strategies - by surface functionalization with targeting ligands and/or payload release via stimuli-responsiveness properties - for OS have been broadly developed, as depicted in Table 1 and 2, respectively.
< Table 1 >
< Table 2 >
3.1. Osteosarcoma-targeted therapies by means of passive targeting Regarding passive targeting, lipid-based nanoparticles, such as liposomes, have been broadly developed to delivery drugs to OS cells. Particularly, doxorubicin was encapsulated in liposomes with natural phospholipids composition, via pH gradient method attaining a final encapsulation efficiency of ca. 84 %[71]. This liposomal doxorubicin (L-Doxorubicin) consists in a spherical vesicle with a hydrophilic core in which doxorubicin was loaded and a lipidic hydrophobic shell, coated with polyethylene glycol (PEG), which improved the stability, increased circulation half-time, diminished uptake by immune cells thus reducing systemic phagocytosis and, therefore, prolonging systemic circulation. Overall, the final liposomes showed an enhanced internalisation of doxorubicin by the OS cell lines and an increase in the cancer cells death, successfully increasing the therapeutic efficacy. PEGylation also facilitates conjugation with other molecules and provides solubility-improving capabilities[6]. Other polymers, such as
bioavailability and consequently the accessibility and accumulation of the nanoparticles to the cancer cells[55]. Several studies can be found in literature reporting the application of nanocarriers to the delivery of different compounds to OS cells, including not only passive but also active targeting purposes[76, 77]. Particularly, a RGD-decorated nanosystem was developed for selective targeting of doxorubicin to the OS cells, showing an improved cellular uptake when compared with the non-targeted nanoparticle. RGD is a
peptide
composed
by
the
amino
acid
sequence
Arg-Gly-Asp
(RGD),
arginylglycylaspartic acid capable of interacting with abundantly expressed surface integrins[78]. Thus, this peptidic ligand was used to specifically target the cancer cells, displaying good features concerning its biocompatibility, safety and targeting abilities. Hence, the nanosystem composed by a biodegradable polymeric micelle loaded with doxorubicin, further decorated with ligands exhibiting high affinity for surface integrins of the cancerous cells (RGD-DOX-PM) showed selective targeting therapy of OS cells, typifying a micellar targeted delivery system with reported selective killing of the malignant cells[63]. Stimuli-responsive conjugated micelles loaded with doxorubicin were designed with acid-sensibility properties due to an acid cleavable hydrazone bond[60], which enabled the selective release of the drug under the well-known tumor microenvironment acidic conditions widely referred in literature[79, 80]. Several physiopathological hallmarks, such as hypoxia, angiogenesis, and a shift from the oxygen-dependent energy production pathways lead to the production of acidic intermediaries, therefore playing a key role in the generation of an acidic extracellular milieu (decrease in pH) not only around the cancer cells but also in the vicinities of noncancerous cells[81]. D-aspartic acid octapeptide has a dual function as micellar component; it serves both as the hydrophilic moiety of the micellar structure and as a ligand for the ligand-directed targeted nanodrug delivery system. pH-sensitive hydrazone
bonds have been broadly used as a strategy in pH-responsive drug delivery cancer nanomedicines[82]. These pH-triggered drug release properties have been extensively applied to a broad set of nanosystems for cancer therapy, including cancer immunotherapy polymeric nanoparticles[83], PEGylated liposomes for chemotherapeutic drug delivery[84], and nanoparticles featuring acid-cleavable maleic acid amides, known as TACMAA[85]. Photodynamic therapy using nanoparticles has been also applied to cancer therapy. There are reports on upconverting nanoparticles containing a photosensitiser agent for breast cancer therapy[86], hollow gold-mitoxantrone PEGylated nanoparticles with the dual function of chemotherapy agent delivery and photodynamic therapy[87], or Fe3O4 and gold-based nanoparticles for theranostics, combining bioimaging and photodynamic therapy[88]. A recent study showed the application of photodynamic therapy as a strategy for OS therapy, using zinc phthalocyanine as the photosensitizer molecule with both photo-physical and photo-chemical attributes. The need for an efficient nanosystem for the loading of the photosensitiser molecule was underlined, owing to the low solubility and crystallization tendencies of the molecule, which could pose a threat to its functional activity[74]. With this purpose in mind, the molecule was incorporated in a PEG-PMAN (amphiphilic block copolymer) polymeric nanoparticle named PEG-PMAN/ZnPc (PPZ), improving the generation of ROS in OS cell lines, as well as cell cycle arrest followed by apoptosis, once light was irradiated and the structure hence activated. This dramatically enhanced the cytotoxicity properties of the nanosystem both in cell cultures and in-vivo experiments[56]. Hyaluronic acid coated cationic liposomes were developed for specific CD-44-targeting, delivering doxorubicin into the OS cells using an interesting intracellular redox-sensible glutathione-triggered drug delivery system via bio-reducible disulfide linker, connecting cholesterol with detachable polyethyleneglycol units
(PEG2000). Nanomedicines featuring redox-sensitive responsive linkers are well documented in literature, such as the case of breast cancer[89] and lung cancer[90, 91]. Viral vectors, such as adenovirus, have been used for OS targeting. This time, a siRNA targeting ezrin was carried in a adenovirus, transported to the human OS MG-63 cells, with interesting results: certain gene levels were augmented, namely Bax, p21, p53, Cyclin D1,Caspase-3, CDK4, whereas others - Bcl-2, metalloproteinases 2 (MMP-2) and 9 (MMP-9) had their expression levels reduced[92]. Cell viability, growth, invasion and migration processes all suffered inhibition after this adenovirus-siRNA ezrin-targeted delivered the inhibitory ribonucleotides, attesting the role of viral vectors for siRNA therapeutic strategies. 3.2. Active targeting to Osteosarcoma cells: specific ligands The development of successful therapeutic strategies for cancer has been notably improved with nanocarriers decorated with surface-bonded molecules of different nature, such as ligands, antibodies and aptamers, for directly guiding to the specific target-cells [93-95]. Most nanocarriers rely solely on the EPR (enhanced permeability and retention) effect, a widely-known passive targeting way in which the specific tumoral milieu can favour the accumulation of nanodrug delivery systems, through increased angiogenesis, diminished lymphatic drainage and a leakage-favouring disorganized architecture of the tumoral tissue [96]. Nevertheless, active targeting emerges as an useful way for enhancing cellular uptake although it has its own challenges since the design of active targeted nanocarriers require sufficient knowledge on the specific tumor cells to be addressed and the nano-bio interactions that can be established with the targeted and non-targeted entities [97]. Overexpression of certain surface cell receptors on cancer cells has revealed to be a promising tool for the development of active targeted drug delivery nanosystems. Folic acid receptors and transferrin receptors are two of the most studied receptors in what
concerns active ligand-targeting nanodelivery systems as a cancer therapy strategy, targeted by the respective ligands: folic acid and transferrin, respectively[98]. Regarding OS, folate entrance into the cancerous cell has been evaluated through the two main mechanisms: folate receptor and reduced folate carrier. Only, folate receptor alpha was overexpressed in OS cancer cell lines but also it was shown that the general folate uptake pathways could play major roles on altering cancer cells’ sensitivity to chemotherapeutic drugs, the development of chemoresistance and the ultimate response to anticancer therapeutics[99-102]. Other relevant targets in OS are ephrin receptors[103], VEGF[104], uPA[105, 106] and ezrin [15, 107]. Recently, ligand-directed toxin for three types of cancer, including OS, was designed. This recombinant protein called eBAT, epidermal growth factor bispecific angiotoxin, showed active targeting towards the epidermal growth factor receptor (EGFR) and the urokinase plasminogen activator receptor (uPAR). In addition to its ability to increase the tumor binding affinity, eBAT also stimulates a cytotoxic response in EGFR and uPAR overexpressing cancer cells. Comparing to the monospecific toxins, the bispecific eBAT showed increased cytotoxicity for the same concentration values, i.e. lower IC50 [108]. An OS-targeted drug delivery system was also recently developed with ligand CD80 and VEGF antibody attached to the magnetic iron oxide nanoparticles. This way, this conjugate system would be able to, not only target OS cells widely-expressed VEGF antigen, which plays major roles in tumor angiogenesis processes, but also the surface cell receptor Cytotoxic T lymphocyte-associated antigen-4 (CTLA4) expressed also by OS cancer cells, in a synergistic attempt for potentiating OS cell death[61]. The VEGFCD80 conjugated nanosystems showed, particularly at 1 µg/mL, the greatest OS cell death efficacy and, after 72 h and three nanoparticle treatments, the CD80+VEGF conjugated nanoparticles showed lower live cell count when compared to the ones only
conjugated with VEGF or CD80. The possible role of the interaction between CD80 ligand and the CTLA-4 receptor, widely known to induce cell death and one of the major standard points in immunotherapy, was also mentioned as a contributor to the results[109]. Moreover, the substantive VEGF receptor expression on OS cells associated with an overexpression of certain genes related to its molecular pathway makes this conjugated nanosystem an interesting strategy for selective targeting and drug delivery to VEGF receptor overexpressing OS cells[110-112]. CD133 aptamers were conjugated with PEGylated poly(lactic-co-glycolic acid) nanoparticles loaded with salinomycin with 50% encapsulation efficiency, for OS cancer stem cells targeting[62]. Cluster of differentiation 133 (CD133) is regarded as a cancer stem cells marker, not only in the case of OS[113] but also in other tumors[114, 115]. The salinomycin-conjugated polymeric nanoparticles (Ap-SAL-NP) were capable of reducing in vivo both tumor volume and tumor weight, when compared to the other formulations (free salinomycin, salinomycin nanoparticles, aptamers-conjugated nanoparticles). At day 60, and comparing to the initial tumoral volume (day 0) the mice treated with free salinomicyn showed a 17.4 times higher tumoral volume, whereas the ones treated with the salinomycin nanoparticles presented 14.2 times higher tumoral volume, contrasting with the aptamer-conjugated salinomycin nanoparticles, this time displaying a 7.1 fold increase in tumoral volume. Selective killing of CD133+ cancer stem cells of OS was obtained as a result, thus showing the future potential of this nanosystem in specific targeting and in cancer therapeutics. In a subsequent study, both the cancer stem cells and the cancer cells were targeted with polymeric-lipid nanoparticles loaded with salinomycin, this time with a dual ligand strategy (EGFR aptamers and the previously used CD133 aptamers), showing interesting results once the cytotoxicity (in two different OS cell lines) of the nanoparticles containing both ligands was superior when compared
to the non-targeted (absence of ligand) or single-targeted (only one ligand) cytotoxic effect, this way representing a better and refined strategy not for only OS cells targeting but also – and synergistically – cancer stem cells targeting[66]. The αvβ3 and αvβ5 integrins have been targeted by Arg-Gly-Asp (RGD) peptide via polymeric micelles composed of amphiphilic block copolymer poly(ethylene glycol)block - poly (trimethylene carbonate) loaded with doxorubicin. RGD-DOX-PM has shown substantial potential as a OS targeting strategy, taking in mind the in vitro results of effective cancer cell killing by the integrin-mediated interactions between the peptide and the cancer cells, as previously described[63].The targeted RGD-DOX polymeric micelles showed a 5.4 fold lower IC50 when compared to the non-targeted control DOXPM in MG-63 cells; this way exhibiting a much higher antitumor activity due to active targeting. Doxorubicin-loaded liposomes were developed in a recent study, this time conjugated with an Ephrin Alpha 2 receptor - targeting peptide called YSA. [51] Ephrins are an extensive family of tyrosine kinase receptors, with functions in cancer proliferation and metastasis well documented, emerging as interesting potential targets for cancer therapy strategies[116, 117]. A couple of studies focusing on ephrin receptors targeting peptides showed the potentiality of ephrin receptors-targeted nanodrug delivery systems [118, 119]. Hence, liposomes were composed by dipalmitoylphosphatidylcholine (DPPC),
cholesterol,
and
polyethyleneglycol-conjugated
distearylphosphatidylethanolamine (DSPE-mPEG), to which the YSA-peptide was conjugated to. Results suggested an increase in specificity and toxicity for this targeted delivery system, when compared to the doxorubicin-free and non-targeted L-doxorubicin, once cytotoxicity to the SAOS cells of the YSA-L-doxorubicin nanosystem was 1.91 fold higher compared to the doxorubicin-free and 1.50 fold higher compared to the non-
targeted counterpart [51]. Nevertheless, OS therapy, as for plenty of other cancers, has major issues regarding chemoresistance [120]. Multidrug resistance (MDR) is a major threat to cancer therapy, as cancer cells become resistant to chemotherapy agents, predominantly by two mechanisms: increased drug efflux transporters on cell membranes, which decreases the intracellular concentration of the drug and by increasing anti-apoptotic signalling pathways [45, 121]. Hence, the codelivery of drugs and nucleic acids, the latter either improving intracellular concentration of chemotherapy agent or activating apoptotic pathways leading to cancer cells apoptosis, can effectively overcome resistance of cancer cells and improve therapy results. Moreover, recent studies reported drug resistance in OS treatment related to long noncoding RNAs (lncRNAs)[122], LIM kinase 1 overexpression[123], circular RNAs (cRNAs)[124], to list a few. Following the ever-increasing importance of this serious issue, a recent study was conducted for dual-targeting of OS, building on previous work, with EphA2 ligand-surface conjugated functionalized cationic liposomes, and intracellular targeting of JNK-interacting protein 1, by siRNA JIP1(Figure 3. A) [67]. Decreased gene expression of JIP1 (46%) and JIP1 mRNA (42%), the latter the target of the JIP1 siRNA, was achieved, and co-delivery of chemotherapeutic drug doxorubicin and gene-silencing JIP1 siRNA showed interesting results in OS cells killing (apoptotic effect), the greatest for the YSA-L-siRNA-DOX active targeted micelleplexes (Figure 3. B). This YSA-L-siRNA-DOX nanosystem has proved to be an interesting strategy for tackling tumor cells and for increasing chemosensitivity within the multidrug resistance (MDR) panorama.
< Figure 3> Figure 3. A) Schematic illustration of the YSA peptide-targeted liposomal DOX-siRNA nanosystem. Nanovesicles were prepared with cholesterol, DPPC, DOTAP, and DSPE–mPEG via pH gradient method.
YSA peptide was conjugated to the liposome as surface modification to target OS cells specifically, using NHS linker strategy. DOX was loaded onto the liposome, whereas siRNA was loaded on the surface of the nanovesicles. Severeal characterization tests and biological assays were carried out to assess the capabilities of the prepared codelivery system. Abbreviations: DOTAP, N-[1-(2,3-dioleoyloxy)propyl]-N,N,Ntrimethylammonium
methyl-sulfate;
DOX,
doxorubicin;
DPPC,
1,2-dipalmitoyl-sn-glycero-3-
phosphocholine; DSPE–mPEG, distearoyl-phosphatidylethanolamine–methyl-poly(ethylene glycol); OS, osteosarcoma. B) OS cell apoptosis by Annexin. Cells were treated with YSA-L, YSA-L-siRNA, DOX, YSA-L-DOX, L-siRNA-DOX, and YSA-L-siRNA-DOX for 48 h. Mean±SD are determined. The symbol * represents a statistically significant difference (P, 0.05) when compared with control. Abbreviations: DOX, doxorubicin; OS, osteosarcoma. Reproduced with permission from [135], copyright by Dovepress.
Polymer-coated folate-targeted gold nanorods (GNRs) were developed for both photothermal and chemotherapy of OS. The two different gold nanorods developed for anticancer drug nutlin-3 delivery were coated with different cytocompatible polymer derivatives: an inulin-based graft copolymer, conjugated with folic acid molecules -INULA-PEG-FA
–
and
α,β-poly(N-2-hydroxyethyl)-DL-aspartamide
(PHEA)
also
conjugated to folic acid - PHEA-EDA-FA. Hyperthermic effect was evaluated in vitro for the two nanosystems and both the nanoparticulate systems demonstrated interesting capabilities for hyperthermic therapy tumor ablation, after NIR laser exposure[64]. Selective tumor accumulation mediated by folic acid targeting was shown on folate receptors-overexpressing U2OS cancer cell lines. Testing in 3D OS models was also done both for chemotherapeutic and hyperthermic therapies, and the results showed that this two active-targeting gold nanorods are interesting strategies for, not only OS chemotherapy but also for photothermal therapy, the latter also extensively referred in literature[125, 126], combining drug molecules and thermic effects for synergistic tumor targeting and its eradication[64]. The utility of gold nanorods as mediators in
photothermic therapy has long been described; their absorptive capacities, stability, biocompatibility and facile biofunctionalization make gold nanorods to be a promising cancer-applied photothermal therapeutic nanomaterial. Other recent study regarding photothermal therapy in the context of OS was developed this time by coating the gold nanorods with polyacrylic acid (PAA), and photothermic therapy through hyperthermic effect was studied in MG63 human OS cell[57]. Apoptosismediated killing of MG63 OS cells, mainly via combination of DNA integration phenomenon and cell membrane disruption showed the notability of this nanosystem for cancer-targeting and photothermal therapy. These nanostructures can be integrated in bioscaffolds for bone repair, thus joining, both the osteogenic effect of the biomaterials and the chemo-photothermal therapeutic synergic effect of this magnetic mesoporous calcium silicate/chitosan nanoplatform, via doxorubicin anticancer effect and tumorreducing photothermic effect[127]. A different approach relies on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas 9 genome editing technology applied to tumor-targeting strategies[128], in which a Vascular Endothelial Growth Factor A (VEGFA) guide RNA (gRNA) was developed for tackling tumor growth and angiogenesis mediated by this type of VEGF overexpressed in OS cells[65]. An aptamer specific for OS cells targeting named LC09 was developed and functionalized to a lipopolymer composed by polyethyleneimine (PEI), methoxypolyethyleneglycol (PEG) and cholesterol (CHOL). CRISPR/ CAS 9 plasmids encoding for the referred guide RNA were loaded into the lipopolymeric structure (LC09-PPC-CRISPR/Cas9 nanosystem) and specific targeting was achieved, as well as a genome editing in the portion of the genome codifying to the VEGFA, resulting not only in diminished tumor growth, but also an inhibition of lung metastasis and angiogenic processes, in orthotopic mouse models for OS and lung lesion-metastatic OS tissue[65, 129]. Other studies regarding CRISPR/Cas9-
mediated targeting of OS can be found, namely ligand 1, for programmed cell death of OS cells[130] and as a drug resistance management strategy in chemoresistant OS cells[131]. 3.3. Micelleplexes for smart delivery of nucleic acids and chemotherapy to osteosarcoma As explained above, micelleplexes refer to stable complexes of genetic material with cationic amphiphilic block copolymers that adopt a micelle-like structure[7]. Nucleic acids, such as plasmidic DNA (pDNA)[132], small interfering RNA (siRNA)[133] and micro RNA (miRNA)[134] have been successfully loaded into cancer-targeted micelleplexes, recently. Combination of nucleic acid delivery with chemotherapy agents aiming synergistic anticancer action has been also explored in a wide variety of cancers[135]. Henceforth, special attention will be given to the major RNA interference (RNAi) agents advances in osteosarcoma pathological framework, as well as the exploitation of cancer-targeted micelleplexes as suitable nanocarriers for nucleic acid delivery to the OS tumor microenvironment. 3.3.1. RNA interference agents in osteosarcoma An interesting strategy developed to a great extent in several diseases is undoubtedly the delivery of small ribonucleic acid molecules with inhibitory properties[136]. Ribonucleic acid (RNA) -based therapeutics have witnessed a huge growth and a large increase in scope in the past years, following the discovery of RNA with catalytic properties in the 80’s and the work of scientists in the late 90’s, culminating in the 2006 Nobel Prize win, for the RNA interference discovery[137]. The potential use of RNA molecules as therapeutic molecules has prompted intensive studies on different types of RNA and their different activities, attesting the versatility of the different ribonucleic acid
molecules[138]. A plethora of RNA molecules have assumed relevance in the landscape of RNA-based therapeutics, such as RNA interference agents – miRNAs, siRNAs, shRNAs-, ribozymes, aptamers, antisense oligonucleotides. Antisense oligonucleotides are single-stranded non-coding sequences of a small number of nucleotides which are specific to certain sequences of mRNA, resulting in the inhibition of the mRNA transcript after complementary binding, consequently affecting gene expression. RNA interference is a dynamic cellular process mediating silencing gene expression, most notoriously posttranscriptional gene silencing processes, in which small RNA molecules play a key role, such as endogenous microRNAs (miRNAs), functioning either as translation repressors or mRNA degradation molecules, after limited complementarity with the mRNA strand, and exogenous small interfering RNAs (siRNAs), serving as mRNA cleaving molecules after base pairing. Indeed, miRNA dysregulation has been implied in cancer progression, and the two main miRNA-based therapeutics consist in restoration of tumor suppressive miRNAs, by administration of miRNA mimics or miRNA-coding genes, or by oncogenic miRNA ablation, recurring to antisense oligonucleotides (ASO) or miRNA sponges, to name a few[139]. As for siRNA, the ability to downregulate expression of molecular targets in different signalling pathways has paved the way to siRNA-based potential therapeutics, currently under extensive investigation either alone or in combination with chemotherapy agents to maximize therapeutic outcome[140]. Despite both miRNA and siRNA-mediated gene silencing mechanisms share the same cytosolic silencing complex – RNA-induced silencing complex (RISC), further exerting their functions regarding translation repression, siRNAs have a higher complementary relationship to the target mRNA and in this way these oligonucleotides are more specific in terms of base pairing, whereas miRNAs are less specific and have a lower degree of complementarity with the targeted RNA messenger molecule[141]. RNAi therapy, in particular siRNA therapy has
been described as advantageous when compared to the traditional drug research and therapy undertones; namely, quicker research and development of specific gene inhibitors, thus less time-consuming; and both potency and selectivity are improved due to the complementarity of the target oligonucleotides to the specific mRNA strand, as opposed to conventional chemotherapy [142]. Focusing on RNAi therapeutics and the delivery of therapeutic oligonucleotides, several challenges can be easily traced, bearing in mind that the integrity of the structure is needed and the advantageous use of nanotechnology and nanocarriers as platforms for miRNA and siRNA delivery. Due to their small size and molecular weight, their renal clearance can be augmented as they are easily excreted via urine; protein adsorption and opsonisation of positive charged RNAi nanocarriers is another aspect to take in mind, resulting in the development of new polymeric coatings to overcome unspecific binding and opsonisation processes. Filtration of RNAi-containing nanoparticles by the reticuloendothelial system (RES) is important, and the physicochemical characteristics of the nanocarriers (shape, size, surface charge, hydrophilicity) are crucial for the final outcome. Large sizes, for example, would increase the opsonisation and uptake process by the RES cells; thus the importance of polymeric molecules capable of reducing or at least limiting RES uptake[143]. The nanoparticle should also reach the target tissue, therefore it must trespass tumoral extracellular matrix and this way reaching the cancerous cells. Despite the abnormal proliferation and secretion of certain cells and contents, accumulation of the nanoparticles in the tumour microenvironment is facilitated due to the vascular heterogeneity and discontinuity (EPR effect). Cell membranes need also to be transposed and for these cellular uptake process size and surface charge are pivotal in their roles, once smaller nanoparticles may be preferentially internalized via clathrin-mediated endocytosis, whereas bigger molecules are normally internalized via caveolae-mediated endocytosis (CvME), a clathrin-
independent endocytic pathway. For particles with positive surface charge, internalization is favoured[143]. The ability of loaded nanoparticles to escape the endosomes is another step to consider, for the successful release of miRNA and siRNA molecules, before degradation, via endosome membrane rupture. This endosomal scape is favored for nanocarriers coated with cationic polymers containing protonable amine groups, hence enhancing membrane disruption and facilitating the RNA molecule delivery. Off-target effects can arise, namely due to a partial complementarity of the strands to an unintended target, and are therefore non-desirable, once they can lead to inflammatory responses. These problems could be addressed through nucleobase modifications[144] and recurring to artificial nucleotide analogues with the ability to reduce off-target effects of siRNA[145]. miRNA-based therapies are, in pair with siRNA therapy, promising strategies and have experimented a notable increase namely within the cancer panorama[146]. miRNA have been extensively studied regarding both their pathological and therapeutical role, and the regulation pathways, downregulation (underexpression), upregulation (overexpression) in cancer cells, this way arising the classification of oncomiRs if the miRNA has an oncogenic effect, and tumor suppressor miRNAs if the miRNA functions as a tumor suppressor molecule[147]. These small nucleic acid molecules (17-21 nucleotides approximately) have shown to be key-players in the oncological molecular landscape and have been therefore extensively studied in their ability to supress cancer development, proliferation, metastization, opening new avenues for potential therapeutic targets in OS pathology (Table 3). < Table 3 > MiRNAs can be categorized in 5 main groups, for instance as potential prognosis biomarkers, thus important in OS diagnosis[169-172]; microRNAs functioning as oncogenic molecules, the oncomirs[165, 173-175]; tumor suppressor-microRNAs[176-
179]; microRNAs playing a role in chemoresistance promotion [180-183] , as well as active molecules in chemoresistance repression[184-187] (Figure 4). siRNA-based strategies for OS targeting include mutant p53 gene knockdown, by using a p53-specific siRNA [188] and Che-1 gene silencing, leading to decrease expression levels mutant p53 gene which further promotes apoptosis in OS cells[189]. < Figure 4> Figure 4. General view of major microRNAs as key molecules interplaying different roles in the pathological framework of osteosarcoma.
3.3.2. Micelleplexes for nucleic acid delivery into the tumour microenvironment The therapeutic potential of small RNA molecules has received increasing attention in the last decade and successful strategies for the efficient delivery of these molecules have led to the development of smart nanosystems for diagnosis or therapeutic purposes[190]. Micelleplexes have since emerged as an interesting strategy for not only nucleic acid delivery but also nucleic acid-drug combinations, as the hydrophobic core can function as a reservoir for hydrophobic drugs, such as chemotherapy agents[45]. This way, synergistic effects can be achieved through the co-delivery of chemotherapeutic agents and nucleic acids to target simultaneously different cellular mechanisms with overall benefit concerning cancer therapy, via direct killing of cancer cells and MDR circumvention. Also, surface chemistry modification allow ligand conjugation, thus displaying active targeting functionalities (Figure 5)[7, 9].
Figure 5. Illustration of a hypothetical multifunctional micelleplex for dual delivery of drugs and nucleic acids.
The interaction between the charged groups and the small RNA molecules and their accessibility on the surface of the micellar structure are critical factors for efficient nucleic acid complexation[191]. Critical factors for successful nucleic acid delivery are 1) nucleic acid complexation, 2) nucleic acid transportation, 3) cellular uptake and internalization of the nucleic acid molecules, and 4) endosomal escape and cytosolic delivery of the biomolecules (Figure 6).
Figure 6. Micelleplexes have ideal properties for genetic material transport and delivery. Targeting ligands may be attached to the surface allowing an active targeting and an increase in efficiency of the therapeutic drugs in cancer therapy, after internalization and consequent release of the genetic material and drug into the cytosol of cancer cells.
It should be noted that, whilst siRNA and miRNA have their ultimate targets within the cytosol, in the case of pDNA further translocation into the nuclear compartment is mandatory. This way, the nuclear envelope means an additional barrier [192]. So far, micelleplexes have been applied to a few number of cancers, combining nucleic acid material with drug delivery taking advantage of the cationic properties, reported in different studies depicted in Table 4.
< Table 4>
The versatility and functionality of micelleplexes as polycationic nanosystems for genetic material carrying and delivery into cells are attested in different studies, exploring different features such as stimuli-responsiveness (pH), ligand conjugation and active
targeting. Dual pH-responsive PDMA-b-PDPA micelles composed of a hydrophilic shell of poly (2-(dimethylamino)ethyl methacrylate) (PDMA) and an hydrophobic core of poly(2-(diisopropylamino) ethyl methacrylate (PDPA) were tested for siRNA and amphotericin B co-delivery. Encapsulation in the micelleplexes allowed for a successful release of amphotericin B and promotion of endosomal disruption via membrane poration for enhanced siRNA delivery into the intracellular milieu[193]. Moreover, the diblock copolymers caused an increase in osmotic pressure, which favoured endosomal membrane tension and destabilization, resulting on the consequent intracellular delivery of the active substances. PDMA‐b‐PDPA diblock copolymers were also used in a different study regarding the co-delivery of a siRNA molecule and paclitaxel through pHresponsive micelleplexes, which were disassembled when pH reached 6.3. At this pH both the PDMA (already partially protonated) and PDPA were protonated, leading to the dissociation of the nanostructure and to the release of paclitaxel inside the tumor cells[194]. Cancer multidrug resistance is a huge challenge and demands effective strategies to counteract this problem[203]. In this sense, dual loading of the micelleplexes with a chemotherapeutic drug and also with siRNA‐Bcl‐2, a specific Bcl-2 targeting siRNA, enabled successful downregulation of Bcl-2 gene, which may reduce multidrug resistance[194]. Paclitaxel and small interfering RNA co-delivery was also accomplished in another study in which a micelleplex composed of an amphiphilic triblock copolymer poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(2-aminoethyl ethylene phosphate) – mPEG-b-PCL-b-PPEEA was developed. PCL hydrophobicity allows the loading of paclitaxel in the micellar core, and the cationic coating composed by PPEEA confers positive charge for electrostatic interactions with the chosen siRNA. The PEG corona favored the accumulation of the micelleplexes within the tumor site [195]. Acid-
activatable micelleplexes for siRNA delivery have been also combined with photodynamic therapy (PDT). PDPA/OEI-C14/PPa (POP) micelleplexes were prepared with the diblock copolymer poly(ethylene glycol)-block-poly(diisopropanol amino ethyl methacrylate-cohydroxyethyl methacrylate) (PEG-b-P(DPA-co-HEA) and then coloaded with pheophorbide A (PPa) – a photosensitizer, for PDT – co-assembled with PDPA, thus forming PDPA-PPa complexes, and an anti-PD-L1 specific siRNA, for blocking PD-L1-PD-1 interaction, and aiming to impact PD-L1 (inhibitory receptor)expressing immune cells[196]. The cationic polymer 1,2-epoxytetradecane alkylated oligoethyleneimine (OEI-C14) was also added to the structure, co-assembled with the PDPA-PPa complexes, in order to increase the binding affinity to siRNA responsive and also to potentiate the endosomal escape. The acid-responsive nanosystem underwent dissociation under the tumoral acidic microenvironment (Figure 7 - (a)). The POP–PDL1 micelleplexes, loaded with the photosensitizer molecule, successfully mediate a PDT with consequent generation of ROS species (Figure 8 - (e)), besides the immunotherapeutic activity of the siRNA-PD-L1 released into the cytosol after micelleplexes internalization and dissociation (Figure 7 - (b)), resulting in an overall anticancer photodynamic immunotherapy effect. Figure 7. Schematic Illustration of the Acid-Activatable POP/siRNA Micelleplexes for PD-L1 Blocking and Photodynamic Cancer Immunotherapy enhancement, a) Chemical structure of the PPa and siRNA coloaded acid-activatable POP micelleplexes. Dissociation occurs at an acidic microenvironment due to the protonation of the PDPA tertiary amino groups; b) Schematic illustration of the POP–PD-L1 micelleplex mediated photodynamic cancer immunotherapy. ROS generation upon PDT by the micelleplexes induces adaptive immune response, triggering pro-inflammatory cytokine secretion and recruiting tumor infiltrating T cells. RNAi of PD-L1 can further enhances the PDT-induced antitumor immune response.
Specifically, the micelleplexes containing both the PDT agent and the siRNA-PD-L1 displayed the highest inhibition of tumor growth (the lowest values for tumor volume growth - Figure 8 - (a)). Also, the PDT and PD-L1 blockade synergistic approach resulted in considerable apoptosis of the cancer cells, suggesting also the synergistic potential of combining two strategies in one nanosystem, as well as an increased antitumor response as consequence of both PDT and the PD-L1 knock-down (Figure 8 - (c)). Moreover, both tumor recurrence (Figure 8 - (d)) and metastasis inhibition (Figure 8 - (f)) parameters were the lowest for the POP-PD-L1 micelleplexes, thus showing the potentialities of this photosensitizer-loaded micelleplexes for efficient lung cancer therapy.
Figure 8. In vivo PD-L1 KD enhanced photodynamic cancer immunotherapy. (a) Tumor growth inhibition by PDT and PD-L1 KD. The B16-F10 tumor-bearing C57BL/6 mice were intravenously injected with the POP micelleplexes and illuminated with laser (671 nm,t= 2 h) post-injection (n = 6); (b) photographs of B16-F10 tumor-bearing C57BL/6 mice taken at 1 or 7 days post-PD-L1 KD and post-PDT; (c) Images of the TUNEL and H&E staining of the primary tumors at the end of antitumor studies (scale bar: 100 μm); (d) tumor recurrence prevention via PDT and PD-L1 blockade-induced adaptive immune response. B16F10-bearing C57BL/6 mice were intravenously injected with the micelleplexes and treated with PDT for triplicates. 4 days after the last treatment, the primary tumors were resected. Mice were then subcutaneously rechallenged with B16-F10 cells at the day for tumor resection and monitored for tumor regression (1: PBS, 2: POP–NC, 3: POP–NC + laser, 4: POP–PD-L1, 5: POP–PD-L1 + laser) (n = 8); (e) CLSM examination of ROS generation in the lung of B16-F10 metastatic tumor-bearing BALB/c mice; (f) metastasis inhibition via PD-L1 KD enhanced PDT in B16-F10 lung metastatic tumor-bearing BALB/c mice; (g) photographs and (h) H&E staining of the metastatic foci of the B16-F10 tumors (scale bar of 100 μm, n = 6). Reproduced with permission from [202], copyright (2016) by American Chemical Society.
PDT and induced cancer immunotherapy was explored also with other nanosystems[204206]. siRNA-p65 and a cisplatin prodrug were loaded in poly(ethylene glycol)-blockpoly(aminolated methacrylate)
glycidyl
methacrylate)-block-poly(2-(diisopropyl
(PEG-b-PAGA-b-PDPA)-composed
micelleplexes.
amino) The
ethyl triblock
copolymer allows the encapsulation of cisplatin prodrug in the PDPA-based hydrophobic core and siRNA was, similar to other experiments, attached to the cationic surface of the micelleplex (M/Pt(IV)-OC/p65). In this case, the cationic nature of the nanosystem is attributed to PAGA (poly(aminolated glycidyl methacrylate), which also has an important contribution to the therapeutic agenda once it enhances the micelleplex tumor-penetrating abilities (Figure 9.A – (a)). siRNA –p65 was capable of downregulating the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) expression, and, as results showed, it can play an interesting role as a strategy for tumor growth inhibition (Figure 9.B – (a)) and in the suppression of distant lung metastasis in breast cancer, showing the highest performance for the M/Pt(IV)-OC/p65 micelleplexes[197].
Figure 9. A) Schematic illustration of the preparation and delivery of cisplatin prodrug and siRNA from pH-responsive micelleplexes. (a) Pt(IV)-OC (prodrug) was loaded in the hydrophobic core and siRNA was loaded on the intermediate layer of the ph.responsive micelleplexes; (b) Micelleplexes are intracellulary activated due to acidic conditions, followed by their dissociation inside late endosome/lysosome due to PDPA core protonation. Then, cytosolic delivery of both the prodrug and siRNA occurs. B)
The
micelleplex co-loaded with siRNA-p65 and Pt(IV)-OC prodrug significantly inhibited the growth of primary tumor and suppressed lung metastasis: (a) Tumor growth curves in a mouse model bearing 4T1 tumors after treatment with various formulations (the black arrows indicated the injection time points) ; (b) H&E staining of tumor sections. Reproduced with permission from [203], copyright by Ivyspring.
This study clearly highlights the actual relevance of using micelleplexes for co-delivery of hydrophobic-chemotherapy drug molecules and RNA interference technology as an effective combo for oncological therapy purposes. In a recent study, researchers reported on the co-delivery of the anticancer drug camptothecin and TNF-α plasmids, co-loaded in pH-responsive and redox-sensitive micelleplexes composed by multiarmed PEG and diethylenetriamine-grafted
polyaspartate
-
PEG-PAsp
(DET)
–
amphiphilic
copolymers[198]. Redox-sensitive disulfide linkers allowed the conjugation between camptothecin and PAsp (DET) and a cis-aconitic cleavable linker anhydride served as an acid-sensitive PEG-PAsp conjugation molecule. As the targeting ligand, folic acid was conjugated with PEG. Once folic acid is recognized by the folic acid receptors on the cancer cells surface, then folate-mediated endocytosis takes place, exposing the micelleplex to the acidic lysosomal environment, which eventually leads to the cleavage of the acid-sensible linker and consequent degradation of the PEG shell, thereby exposing the policationic PAsp (DET) layer, triggering endosomal escape via proton-sponge effect. Since the cancer cells cytosolic conditions are reductive due to high glutathione (GSH) concentrations, the disulfide bond cleaves, the polyplex is further destabilized and the release of the drug camptothecin occurs, followed by pDNA dissociation [198]. Also recently, folic acid was used as the target molecule conjugated with triblock copolymers - poly-2-methyl-oxazole - poly(dimethylsiloxane)- poly-2-methyl-oxazole (FA–PMOXA–b-PDMS–b-PMOXA–FA)[199]. Pentablock copolymers - poly-2methyl-oxazole (hydrophilic layer) - poly-2-(4-aminobutyl)-oxazole (cationic layer that surrounds the hydrophobic core) - poly(dimethylsiloxane) - poly-2-(4-aminobutyl)oxazole - poly-2-methyl-oxazole (PMOXA–b-PABOXA–b-PDMS–b-PABOXA–bPMOXA) were used this time. Herein, siRNA was loaded into the micelleplexes, attached to the cationic chains of the PMOXA polymer, and results showed promising selective
observed[201]. Therefore, this strategy shows the importance of targeting also the cancer stem cells, giving continuity to the plenty of studies regarding the probable key-role of cancer stem cells in the development, progression, cancer relapse and multidrug resistance features of different types of cancer, such as breast cancer[217], brain cancer[218] and OS[219], to name a few recent works. Theranostic-based nanoformulations (nanotheranostics) have seen a remarkable growth in the past years due to the advantageous “two-in-one” combination of both therapeutic molecules and diagnostic entities [220, 221]. This way, in addition to the gene silencing and chemotherapeutic drug-delivery, it is possible to increase the system’s functionality by adding this diagnostic entity. A micelleplex nanotheranostic platform has been designed for the delivery of siRNA, a drug and a diagnostic imaging agent. Specifically, the cationic copolymer PDMA-block-poly(ε-caprolactone) (PDMA-b-PCL) and mPEGPCL were the chosen copolymers for the SN-38/USPIO-loaded siRNA-PEG micelleplex structure for colon cancer therapy[202]. VEGF siRNA (PEG-conjugated) targets cancer cells human vascular endothelial growth factor (VEGF), SN-38 is the topoisomerase inhibitor anticancer drug 7-ethyl-10-hydroxycamptothecin, and USPIO consists in iron oxide nanoparticles with superparamagnetic properties acting as a contrast agent for bioimaging in magnetic resonance imaging (MRI) technology.
4. Discussion and future directions Despite major advances in the field of cancer therapy and most precisely in OS therapy, there is still major ground to be trilled. A more integrative approach would be very useful, regarding omic approaches, namely proteomic and transcriptomic assays[222, 223]. The role of prognostic markers may need to be further elucidated and investigated; perhaps a multi-combination could be of major interest. Attention should be paid to new and more
effective targets and also novel strategies for selective targeting and with higher drugdelivery efficiency. In this context, new nanoplatforms for the delivery of nucleic acids, chemodrugs, photodynamic therapy, photothermal therapy and immunotherapy should be further improved. The combination of different therapies is also widely-touted to be one of the key factors for therapeutic success in OS and cancer therapy in general, by allying photodynamic therapy with immunotherapy and a chemotherapeutic drug, or even adding a small nuclei acid (miRNA, siRNA), towards more complex but more effective and complementary/ synergistic therapeutic outcomes. Although micelleplexes with tunable positive charges are ideal not only for nucleic acid delivery but also for enhancing cellular uptake, there are some concerns regarding the possible cytotoxicity of the highly positivecharged polymers; thus, further studies are needed in order to elucidate their safety[224, 225]. Theranostic represents a powerful tool for cancer management through the combination of site-specific bioimaging and therapeutics. New advances in polymer chemistry should lead to safer, more proficient biomaterials with biomimetic properties that may render more efficient nanocarriers. The field of biomimetics, cell membranerevested nanoparticles and camouflaged nanoparticles, a relatively recent exciting field of research [226], is gaining increasing attention in cancer-targeting therapy. Numerous advantages play in favour of biomimetic nanocarriers for cargo delivery, namely their functionality,
versatility,
targeting
capabilities,
prolonged
drug
delivery,
biocompatibility. Cell membranes suitable for camouflage can be obtained from different sources, such as cancer cells, red blood cells, white blood cells or platelets[227]. Phototherapy, either photodynamic or photothermal, are one of major therapeutic applications of these nanosystems[228], but also as drug delivery nanocarriers that combine phototherapy with chemotherapy. Hybrid biomimetic membranes can be obtained by fusing membranes of both cancer – melanoma cell line - and red blood cells,
thus taking advantage of the ability of red blood cells to scape immune system and to prolong the circulation time, and the maintenance of specific ligands characteristic of cancer cells, hence consisting in a promising strategy for a cancer targeting improvement[229]. The role of the tumor microenviroment, the interrelationships between the tumor itself and its surroundings as well as the pathologic pathways, are of extreme importance not only to understand better this disease but also for future therapeutic directions[230]. Network-guided controllability analysis could help elucidating new targets for OS therapy, namely in what concerns its tumoral microenvironment[231]. Importance is also given to mesenchymal stem cells as interplayers in the development of the disease[232, 233]. The place of extracellular vesicles in cancer biology has been broadly marked, due to their relevance as potential regulators of cancer development, as key-communicators mediating different interfaces and also as potential therapeutic targets. Despite the need for further developments and a more extensive research, the pathological and the potential therapeutic roles of the OSderived extracellular vesicles (EVs) have already been highlighted. EVs perform as major intercellular mediators, capable of interfering with important OS signalling pathways, namely Wnt/β-catenin and TGF-β ones, and as delivery systems[234]. The role of exosomes as intercellular communicators in OS is not fully understood yet, but their capability to transport different molecules, such as microRNAs, opens new boundaries to the extent and scope of such nanovesicles, emerging as multifaceted players capable of transporting different information[235-237]. Multidrug resistance demands increasing awareness, since it seriously compromises the response to therapy; new developments and advances in this field are needed. Much has been done in trying to understand the role of non-coding molecules, the molecular signalling pathways on display here, showing the utter relevance of a research progress for better therapeutic outcomes. Other
important aspect resides in the ability of a loaded nanosystem to escape and circumvent the natural barriers, hence the necessity of improvements in nanomedicine technology.
Acknowledgements This work was supported by the grant FCT PTDC/CTM-BIO/1518/2014 from the Portuguese Foundation for Science and Technology (FCT) and the European Community Fund (FEDER) through the COMPETE2020 program.This work received financial support from National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência) through project UID/QUI/50006/2013, co-financed by European Union (FEDER under the Partnership Agreement PT2020). It was also supported by the grant FCT PTDC/BTM-MAT/30255/2017 (POCI-010145-FEDER-030255) from the Portuguese Foundation for Science and Technology (FCT) and the European Community Fund (FEDER) through the COMPETE2020 program. This research was also funded by MINECO (SAF2017-83118-R), Agencia Estatal de Investigación (AEI) Spain, Xunta de Galicia (Grupo de Referencia Competitiva ED431C 2016/008), and FEDER (Spain).
References 1. 2. 3. 4. 5. 6. 7. 8.
Siegel, R.L., K.D. Miller, and A. Jemal, Cancer statistics, 2018. CA Cancer J Clin, 2018. 68(1): p. 7-30. Bray, F., et al., Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 2018. 68(6): p. 394-424. Mercatelli, D., et al., Immunoconjugates for Osteosarcoma Therapy: Preclinical Experiences and Future Perspectives. Biomedicines, 2018. 6(1): p. 19. Misaghi, A., et al., Osteosarcoma: a comprehensive review. SICOT-J, 2018. 4. Saraf, A.J., J.M. Fenger, and R.D. Roberts, Osteosarcoma: Accelerating Progress Makes for a Hopeful Future. Front Oncol, 2018. 8: p. 4. Aulton, M.E. and K. Taylor, Aulton's Pharmaceutics: The Design and Manufacture of Medicines. 4th ed. Chapter 45: Pharmaceutical nanotechnology and nanomedicines. 2013: Churchill Livingstone/Elsevier. Amjad, M.W., et al., Recent advances in the design, development, and targeting mechanisms of polymeric micelles for delivery of siRNA in cancer therapy. Prog Polym Sci, 2017. 64: p. 154-181. Kim, H.J., et al., Recent progress in development of siRNA delivery vehicles for cancer therapy. Adv Drug Deliv Rev, 2016. 104: p. 61-77.
Jhaveri, A.M. and V.P. Torchilin, Multifunctional polymeric micelles for delivery of drugs and siRNA. Front Pharmacol, 2014. 5. Zhou, Q., et al., Stimuli-responsive polymeric micelles for drug delivery and cancer therapy. Int J Nanomedicine, 2018. 13: p. 2921-2942. Cai, Y., T. Cai, and Y. Chen, Wnt pathway in osteosarcoma, from oncogenic to therapeutic. J Cell Biochem, 2014. 115(4): p. 625-31. Malumbres, M., Cyclin-dependent kinases. Gen Biol 2014. 15(6). Hughes, D.P., How the NOTCH pathway contributes to the ability of osteosarcoma cells to metastasize. Cancer Treat Res, 2009. 152: p. 479-96. Ren, L. and C. Khanna, Role of ezrin in osteosarcoma metastasis. Adv Exp Med Biol, 2014. 804: p. 181-201. Zhong, G.X., et al., The clinical significance of the Ezrin gene and circulating tumor cells in osteosarcoma. Onco Targets Ther, 2017. 10: p. 527-533. Kleinerman, E.S., Current Advances in Osteosarcoma. 2014: Springer International Publishing. Posthumadeboer, J., et al., Surface proteomic analysis of osteosarcoma identifies EPHA2 as receptor for targeted drug delivery. Br J Cancer, 2013. 109(8): p. 2142-54. Çelik, H., et al., Ezrin Inhibition Up-regulates Stress Response Gene Expression. J Biol Chem, 2016. 291(25): p. 13257-13270. Liu, P., et al., Ezrin/NF-kappaB Pathway Regulates EGF-induced EpithelialMesenchymal Transition (EMT), Metastasis, and Progression of Osteosarcoma. Med Sci Monit, 2018. 24: p. 2098-2108. Rettew, A.N., P.J. Getty, and E.M. Greenfield, Receptor tyrosine kinases in osteosarcoma: not just the usual suspects. Adv Exp Med Biol, 2014. 804: p. 47-66. Tang, M.L., et al., MMP-1 Over-expression Promotes Malignancy and Stem-Like Properties of Human Osteosarcoma MG-63 Cells In Vitro. Curr Med Sci, 2018. 38(5): p. 809-817. Zhang, M. and X. Zhang, Association of MMP-2 expression and prognosis in osteosarcoma patients. Int J Clin Exp Pathol, 2015. 8(11): p. 14965-70. Yi, W.R., et al., Downregulation of IDH2 exacerbates the malignant progression of osteosarcoma cells via increased NF-kappaB and MMP-9 activation. Oncol Rep, 2016. 35(4): p. 2277-85. Berman, S.D., et al., Metastatic osteosarcoma induced by inactivation of Rb and p53 in the osteoblast lineage. Proc Natl Acad Sci USA, 2008. 105(33): p. 11851-11856. Gross, A.C., et al., IL-6 and CXCL8 mediate osteosarcoma-lung interactions critical to metastasis. JCI Insight, 2018. 3(16). Gordon, N. and E.S. Kleinerman, The Role of Fas/FasL in the Metastatic Potential of Osteosarcoma and Targeting this Pathway for the Treatment of Osteosarcoma Lung Metastases, in Pediatric and Adolescent Osteosarcoma, N. Jaffe, O.S. Bruland, and S. Bielack, Editors. 2010, Springer US: Boston, MA. p. 497-508. Huang, G., et al., Participation of the Fas/FasL Signaling Pathway and the Lung Microenvironment in the Development of Osteosarcoma Lung Metastases, in Current Advances in Osteosarcoma, M.D.E.S. Kleinerman, Editor. 2014, Springer International Publishing: Cham. p. 203-217. Zhu, J., et al., Down-Regulation of miR-183 Promotes Migration and Invasion of Osteosarcoma by Targeting Ezrin. Am J Pathol, 2012. 180(6): p. 2440-2451. Yu, X.W., et al., Prognostic significance of VEGF expression in osteosarcoma: a metaanalysis. Tumour Biol, 2014. 35(1): p. 155-60. Kansara, M., et al., Translational biology of osteosarcoma. Nat Rev Cancer, 2014. 14(11): p. 722-35. Society, A.C. Treating Osteosarcoma. 2019 21/06/2019]; Available from: https://www.cancer.org/cancer/osteosarcoma/treating.html.
32. 33. 34. 35. 36. 37. 38. 39. 40.
41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.
Zhang, Y., et al., Progress in the chemotherapeutic treatment of osteosarcoma. Oncol Lett, 2018. 16(5): p. 6228-6237. Lettieri, C.K., et al., Progress and opportunities for immune therapeutics in osteosarcoma. Immunotherapy, 2016. 8(10): p. 1233-44. Miwa, S., et al., Current and Emerging Targets in Immunotherapy for Osteosarcoma. J Oncol 2019. 2019. Wedekind, M.F., L.M. Wagner, and T.P. Cripe, Immunotherapy for osteosarcoma: Where do we go from here? Pediatr Blood Cancer, 2018. 65(9): p. e27227. Lussier, D.M., et al., Combination immunotherapy with alpha-CTLA-4 and alpha-PD-L1 antibody blockade prevents immune escape and leads to complete control of metastatic osteosarcoma. J Immunother Cancer, 2015. 3: p. 21. Koksal, H., et al., Treating osteosarcoma with CAR T cells. Scand J Immunol, 2019. 89(3): p. e12741. Fang, X., C. Jiang, and Q. Xia, Effectiveness evaluation of dendritic cell immunotherapy for osteosarcoma on survival rate and in vitro immune response. Genet Mol Res, 2015. 14(4): p. 11763-70. Kawano, M., et al., Dendritic cells combined with doxorubicin induces immunogenic cell death and exhibits antitumor effects for osteosarcoma. Oncol Lett, 2016. 11(3): p. 2169-2175. Pappo, A.S., et al., A phase 2 trial of R1507, a monoclonal antibody to the insulin-like growth factor-1 receptor (IGF-1R), in patients with recurrent or refractory rhabdomyosarcoma, osteosarcoma, synovial sarcoma, and other soft tissue sarcomas: results of a Sarcoma Alliance for Research Through Collaboration study. Cancer, 2014. 120(16): p. 2448-56. Geller, D.S., et al., Targeted therapy of osteosarcoma with radiolabeled monoclonal antibody to an insulin-like growth factor-2 receptor (IGF2R). Nucl Med Biol, 2016. 43(12): p. 812-817. Zhu, W., et al., Anti-ganglioside GD2 monoclonal antibody synergizes with cisplatin to induce endoplasmic reticulum-associated apoptosis in osteosarcoma cells. Pharmazie, 2018. 73(2): p. 80-86. Gvozdenovic, A., et al., A bispecific antibody targeting IGF-IR and EGFR has tumor and metastasis suppressive activity in an orthotopic xenograft osteosarcoma mouse model. Am J Cancer Res, 2017. 7(7): p. 1435-1449. Liu, Q., et al., MiR-92a Inhibits the Progress of Osteosarcoma Cells and Increases the Cisplatin Sensitivity by Targeting Notch1. Biomed Res Int, 2018. 2018: p. 9870693. Huang, W., et al., Nanomedicine-based combination anticancer therapy between nucleic acids and small-molecular drugs. Adv Drug Deliv Rev, 2017. 115: p. 82-97. Ku, S.H., et al., Chemical and structural modifications of RNAi therapeutics. Adv Drug Deliv Rev, 2016. 104: p. 16-28. Lundstrom, K., Viral Vectors in Gene Therapy. Diseases (Basel, Switzerland), 2018. 6(2): p. 42. Yin, H., et al., Non-viral vectors for gene-based therapy. Nat Rev Genet, 2014. 15: p. 541-555. Nayerossadat, N., T. Maedeh, and P.A. Ali, Viral and nonviral delivery systems for gene delivery. Adv Biomed Res, 2012. 1. Estanqueiro, M., et al., Nanotechnological carriers for cancer chemotherapy: The state of the art. Colloids Surf B: Biointerfaces, 2015. 126: p. 631-648. Haghiralsadat, F., et al., EphA2 Targeted Doxorubicin-Nanoliposomes for Osteosarcoma Treatment. Pharm Res, 2017. 34(12): p. 2891-2900. He, G., et al., Cross Talk Between Autophagy and Apoptosis Contributes to ZnO Nanoparticle-Induced Human Osteosarcoma Cell Death. Adv Healthc Mater, 2018. 7(17): p. e1800332.
53. 54. 55. 56. 57. 58. 59. 60. 61.
62. 63. 64. 65. 66. 67. 68. 69.
Zhang, L., et al., Polymeric nanoparticle-based delivery of microRNA-199a-3p inhibits proliferation and growth of osteosarcoma cells. Int J Nanomedicine, 2015. 10: p. 291324. Wang, B., et al., Paclitaxel and etoposide co-loaded polymeric nanoparticles for the effective combination therapy against human osteosarcoma. J Nanobiotechnol, 2015. 13: p. 22. Wang, L., et al., The effective combination therapy against human osteosarcoma: doxorubicin plus curcumin co-encapsulated lipid-coated polymeric nanoparticulate drug delivery system. Drug Deliv, 2016. 23(9): p. 3200-3208. Yu, W., et al., Zinc phthalocyanine encapsulated in polymer micelles as a potent photosensitizer for the photodynamic therapy of osteosarcoma. Nanomedicine, 2018. 14(4): p. 1099-1110. Pan, S., et al., The Effect of Photothermal Therapy on Osteosarcoma With Polyacrylic Acid-Coated Gold Nanorods. Dose Response, 2018. 16(3): p. 1559325818789841. Prasad, S.R., T.S.S. Kumar, and A. Jayakrishnan, Ceramic core with polymer corona hybrid nanocarrier for the treatment of osteosarcoma with co-delivery of protein and anti-cancer drug. Nanotechnology, 2018. 29(1): p. 015101. Zhou, Z.F., et al., Calcium phosphate-phosphorylated adenosine hybrid microspheres for anti-osteosarcoma drug delivery and osteogenic differentiation. Biomaterials, 2017. 121: p. 1-14. Low, S.A., J. Yang, and J. Kopecek, Bone-targeted acid-sensitive doxorubicin conjugate micelles as potential osteosarcoma therapeutics. Bioconjug Chem, 2014. 25(11): p. 2012-20. Kovach, A.K., et al., Prospective Preliminary In Vitro Investigation of a Magnetic Iron Oxide Nanoparticle Conjugated with Ligand CD80 and VEGF Antibody As a Targeted Drug Delivery System for the Induction of Cell Death in Rodent Osteosarcoma Cells. Biores Open Access, 2016. 5(1): p. 299-307. Ni, M., et al., Poly(lactic-co-glycolic acid) nanoparticles conjugated with CD133 aptamers for targeted salinomycin delivery to CD133+ osteosarcoma cancer stem cells. Int J Nanomedicine, 2015. 10: p. 2537-54. Fang, Z., et al., Targeted osteosarcoma chemotherapy using RGD peptide-installed doxorubicin-loaded biodegradable polymeric micelle. Biomed Pharmacother, 2017. 85: p. 160-168. Li Volsi, A., et al., Near-Infrared Light Responsive Folate Targeted Gold Nanorods for Combined Photothermal-Chemotherapy of Osteosarcoma. ACS Appl Mater Interfaces, 2017. 9(16): p. 14453-14469. Liang, C., et al., Tumor cell-targeted delivery of CRISPR/Cas9 by aptamer-functionalized lipopolymer for therapeutic genome editing of VEGFA in osteosarcoma. Biomaterials, 2017. 147: p. 68-85. Chen, F., et al., Targeted salinomycin delivery with EGFR and CD133 aptamers based dual-ligand lipid-polymer nanoparticles to both osteosarcoma cells and cancer stem cells. Nanomedicine, 2018. 14(7): p. 2115-2127. Haghiralsadat, F., et al., Codelivery of doxorubicin and JIP1 siRNA with novel EphA2targeted PEGylated cationic nanoliposomes to overcome osteosarcoma multidrug resistance. Int J Nanomedicine, 2018. 13: p. 3853-3866. Meshkini, A. and H. Oveisi, Methotrexate-F127 conjugated mesoporous zinc hydroxyapatite as an efficient drug delivery system for overcoming chemotherapy resistance in osteosarcoma cells. Colloids Surf B Biointerfaces, 2017. 158: p. 319-330. Li, L., et al., Association of anti-HER2 antibody with graphene oxide for curative treatment of osteosarcoma. Nanomedicine, 2018. 14(2): p. 581-593.
Chi, Y., et al., Redox-sensitive and hyaluronic acid functionalized liposomes for cytoplasmic drug delivery to osteosarcoma in animal models. J Control Release, 2017. 261: p. 113-125. Haghiralsadat, F., et al., A Novel Approach on Drug Delivery: Investigation of A New Nano-Formulation of Liposomal Doxorubicin and Biological Evaluation of Entrapped Doxorubicin on Various Osteosarcoma Cell Lines. Cell J, 2017. 19(Suppl 1): p. 55-65. Hanafy, N.A.N., M. El-Kemary, and S. Leporatti, Micelles Structure Development as a Strategy to Improve Smart Cancer Therapy. Cancers (Basel), 2018. 10(7). Syu, W.-J., et al., Improved Photodynamic Cancer Treatment by Folate-Conjugated Polymeric Micelles in a KB Xenografted Animal Model. Small, 2012. 8(13): p. 20602069. Pan, S., et al., The Effect of Photothermal Therapy on Osteosarcoma With Polyacrylic Acid–Coated Gold Nanorods. Dose-Response, 2018. 16: p. 155932581878984. Zhou, Z.-F., et al., Calcium phosphate-phosphorylated adenosine hybrid microspheres for anti-osteosarcoma drug delivery and osteogenic differentiation. Biomaterials, 2017. 121: p. 1-14. González-Fernández, Y., et al., Oral administration of edelfosine encapsulated lipid nanoparticles causes regression of lung metastases in pre-clinical models of osteosarcoma. Cancer Lett, 2018. 430: p. 193-200. Li, S., et al., Sarcoma-Targeting Peptide-Decorated Polypeptide Nanogel Intracellularly Delivers Shikonin for Upregulated Osteosarcoma Necroptosis and Diminished Pulmonary Metastasis. Theranostics, 2018. 8(5): p. 1361-1375. Sun, Y., et al., RGD Peptide-Based Target Drug Delivery of Doxorubicin Nanomedicine. Drug Dev Res, 2017. 78(6): p. 283-291. Li, W. and X. Sun, Recent Advances In Developing Novel Anti-Cancer Drugs Targeting Tumor Hypoxic and Acidic Microenvironments. Recent Pat Anticancer Drug Discov, 2018. 13(4): p. 455-468. Kobayashi, H., Cancer Chemotherapy Specific to Acidic Nests. Cancers (Basel), 2017. 9(4). Fernandes, C., D. Suares, and M.C. Yergeri, Tumor Microenvironment Targeted Nanotherapy. Front Pharmacol, 2018. 9: p. 1230. Sui, B., et al., Self-assembled micelles composed of doxorubicin conjugated Y-shaped PEG-poly(glutamic acid)2 copolymers via hydrazone linkers. Molecules, 2014. 19(8): p. 11915-32. Kim, H., et al., Acidic pH-responsive polymer nanoparticles as a TLR7/8 agonist delivery platform for cancer immunotherapy. Nanoscale, 2018. 10(44): p. 20851-20862. Yang, M.M., W.R. Wilson, and Z. Wu, pH-Sensitive PEGylated liposomes for delivery of an acidic dinitrobenzamide mustard prodrug: Pathways of internalization, cellular trafficking and cytotoxicity to cancer cells. Int J Pharm, 2017. 516(1-2): p. 323-333. Du, J.-Z., H.-J. Li, and J. Wang, Tumor-Acidity-Cleavable Maleic Acid Amide (TACMAA): A Powerful Tool for Designing Smart Nanoparticles To Overcome Delivery Barriers in Cancer Nanomedicine. Acc Chem Res, 2018. 51(11): p. 2848-2856. Buchner, M., et al., Photosensitiser functionalised luminescent upconverting nanoparticles for efficient photodynamic therapy of breast cancer cells. Photochem Photobiol Sci, 2019. 18(1): p. 98-109. Imanparast, A., et al., Pegylated hollow gold-mitoxantrone nanoparticles combining photodynamic therapy and chemotherapy of cancer cells. Photodiagnosis Photodyn Ther, 2018. 23: p. 295-305. Rajkumar, S. and M. Prabaharan, Theranostics Based on Iron Oxide and Gold Nanoparticles for Imaging- Guided Photothermal and Photodynamic Therapy of Cancer. Curr Top Med Chem, 2017. 17(16): p. 1858-1871.
Hu, C., et al., Redox-Sensitive Folate-Conjugated Polymeric Nanoparticles for Combined Chemotherapy and Photothermal Therapy Against Breast Cancer. J Biomed Nanotechnol, 2018. 14(12): p. 2018-2030. Song, Y., et al., Paclitaxel-loaded redox-sensitive nanoparticles based on hyaluronic acid-vitamin E succinate conjugates for improved lung cancer treatment. Int J Nanomedicine, 2018. 13: p. 1585-1600. Wang, G., et al., RGD peptide-modified, paclitaxel prodrug-based, dual-drugs loaded, and redox-sensitive lipid-polymer nanoparticles for the enhanced lung cancer therapy. Biomed Pharmacother, 2018. 106: p. 275-284. Tao, Z.W. and P.A. Zou, Adenovirus-mediated small interfering RNA targeting ezrin induces apoptosis and inhibits metastasis of human osteosarcoma MG-63 cells. Biosci Rep, 2018. 38(4). Lee, M.S., E.C. Dees, and A.Z. Wang, Nanoparticle-Delivered Chemotherapy: Old Drugs in New Packages. Oncology (Williston Park), 2017. 31(3): p. 198-208. Muhamad, N., T. Plengsuriyakarn, and K. Na-Bangchang, Application of active targeting nanoparticle delivery system for chemotherapeutic drugs and traditional/herbal medicines in cancer therapy: a systematic review. Int J Nanomedicine, 2018. 13: p. 3921-3935. Bazak, R., et al., Cancer active targeting by nanoparticles: a comprehensive review of literature. J Cancer Res Clin Oncol, 2015. 141(5): p. 769-784. Maeda, H., H. Nakamura, and J. Fang, The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv Drug Deliv Rev, 2013. 65(1): p. 71-9. Rosenblum, D., et al., Progress and challenges towards targeted delivery of cancer therapeutics. Nat Commun, 2018. 9(1): p. 1410. Kolhatkar, R., A. Lote, and H. Khambati, Active tumor targeting of nanomaterials using folic acid, transferrin and integrin receptors. Curr Drug Discov Technol, 2011. 8(3): p. 197-206. Ifergan, I., et al., Reduced folate carrier protein expression in osteosarcoma: implications for the prediction of tumor chemosensitivity. Cancer, 2003. 98(9): p. 195866. Serra, M., et al., Analysis of dihydrofolate reductase and reduced folate carrier gene status in relation to methotrexate resistance in osteosarcoma cells. Ann Oncol, 2004. 15(1): p. 151-60. Yang, R., et al., The folate receptor alpha is frequently overexpressed in osteosarcoma samples and plays a role in the uptake of the physiologic substrate 5methyltetrahydrofolate. Clin Cancer Res, 2007. 13(9): p. 2557-67. Goricar, K., et al., Influence of the folate pathway and transporter polymorphisms on methotrexate treatment outcome in osteosarcoma. Pharmacogenet Genomics, 2014. 24(10): p. 514-21. Guenther, R., et al., Expression of ephrins in human osteosarcoma and chondrosarcoma. Cancer Res, 2006. 66(8 Supplement): p. 274-274. Peng, N., et al., Silencing of VEGF inhibits human osteosarcoma angiogenesis and promotes cell apoptosis via VEGF/PI3K/AKT signaling pathway. Am J Transl Res, 2016. 8(2): p. 1005-15. Ingangi, V., et al., The urokinase receptor-derived cyclic peptide [SRSRY] suppresses neovascularization and intravasation of osteosarcoma and chondrosarcoma cells. Oncotarget, 2016. 7(34): p. 54474-54487. Endo-Munoz, L., et al., Progression of Osteosarcoma from a Non-Metastatic to a Metastatic Phenotype Is Causally Associated with Activation of an Autocrine and Paracrine uPA Axis. PLoS One, 2015. 10(8): p. e0133592.
Zhang, Y., et al., Osteosarcoma metastasis: prospective role of ezrin. Tumour Biol, 2014. 35(6): p. 5055-9. Oh, F., et al., Targeting EGFR and uPAR on human rhabdomyosarcoma, osteosarcoma, and ovarian adenocarcinoma with a bispecific ligand-directed toxin. Clin Pharmacol, 2018. 10: p. 113-121. Sharma, A., et al., Anti-CTLA-4 immunotherapy does not deplete FOXP3+ regulatory T cells (Tregs) in human cancers. Clin Cancer Res, 2018. Bajpai, J., et al., VEGF expression as a prognostic marker in osteosarcoma. Pediatr Blood Cancer, 2009. 53(6): p. 1035-9. Oda, Y., et al., CXCR4 and VEGF expression in the primary site and the metastatic site of human osteosarcoma: analysis within a group of patients, all of whom developed lung metastasis. Mod Pathol, 2006. 19(5): p. 738-45. Liu, Y., et al., High expression levels of Cyr61 and VEGF are associated with poor prognosis in osteosarcoma. Pathol Res Pract, 2017. 213(8): p. 895-899. Tirino, V., et al., Human primary bone sarcomas contain CD133+ cancer stem cells displaying high tumorigenicity in vivo. FASEB J, 2011. 25(6): p. 2022-30. Kazama, S., et al., Expression of the stem cell marker CD133 is related to tumor development in colorectal carcinogenesis. Asian J Surg, 2018. 41(3): p. 274-278. Sui, Y.P., et al., Prognostic value of cancer stem cell marker CD133 expression in esophageal carcinoma: A meta-analysis. Mol Clin Oncol, 2016. 4(1): p. 77-82. Lodola, A., et al., Targeting Eph/ephrin system in cancer therapy. Eur J Med Chem, 2017. 142: p. 152-162. Saha, N., et al., Therapeutic potential of targeting the Eph/ephrin signaling complex. Int J Biochem Cell Biol, 2018. 105: p. 123-133. Riedl, S.J. and E.B. Pasquale, Targeting the Eph System with Peptides and Peptide Conjugates. Curr Drug Targets, 2015. 16(10): p. 1031-1047. Ma, B., et al., Investigation of the interactions between the EphB2 receptor and SNEW peptide variants. Growth factors (Chur, Switzerland), 2014. 32(6): p. 236-246. Yang, J.Z., et al., Characterization of multidrugresistant osteosarcoma sublines and the molecular mechanisms of resistance. Mol Med Rep, 2016. 14(4): p. 3269-76. Creixell, M. and N.A. Peppas, Co-delivery of siRNA and therapeutic agents using nanocarriers to overcome cancer resistance. Nano Today, 2012. 7(4): p. 367-379. Han, Z. and L. Shi, Long non-coding RNA LUCAT1 modulates methotrexate resistance in osteosarcoma via miR-200c/ABCB1 axis. Biochem Biophys Res Commun, 2018. 495(1): p. 947-953. Yang, J.Z., et al., LIM kinase 1 serves an important role in the multidrug resistance of osteosarcoma cells. Oncol Lett, 2018. 15(1): p. 250-256. Kun-Peng, Z., et al., Screening circular RNA related to chemotherapeutic resistance in osteosarcoma by RNA sequencing. Epigenomics, 2018. 10(10): p. 1327-1346. Grosges, T. and D. Barchiesi, Gold Nanoparticles as a Photothermal Agent in Cancer Therapy: The Thermal Ablation Characteristic Length. Molecules, 2018. 23(6). Wu, Y., et al., Gold Nanorod Photothermal Therapy Alters Cell Junctions and Actin Network in Inhibiting Cancer Cell Collective Migration. ACS Nano, 2018. 12(9): p. 92799290. Yang, F., et al., Magnetic Mesoporous Calcium Sillicate/Chitosan Porous Scaffolds for Enhanced Bone Regeneration and Photothermal-Chemotherapy of Osteosarcoma. Sci Rep, 2018. 8(1): p. 7345. Martinez-Lage, M., et al., CRISPR/Cas9 for Cancer Therapy: Hopes and Challenges. Biomedicines, 2018. 6(4). Liu, T., et al., CRISPR-Cas9-Mediated Silencing of CD44 in Human Highly Metastatic Osteosarcoma Cells. Cell Physiol Biochem, 2018. 46(3): p. 1218-1230.
Liao, Y., et al., Targeting programmed cell death ligand 1 by CRISPR/Cas9 in osteosarcoma cells. Oncotarget, 2017. 8(18): p. 30276-30287. Liu, T., et al., Targeting ABCB1 (MDR1) in multi-drug resistant osteosarcoma cells using the CRISPR-Cas9 system to reverse drug resistance. Oncotarget, 2016. 7(50): p. 8350283513. Kara, A., et al., Development of novel self-assembled polymeric micelles from partially hydrolysed poly(2-ethyl-2-oxazoline)-co-PEI-b-PCL block copolymer as non-viral vectors for plasmid DNA in vitro transfection. Artif Cells Nanomed Biotechnol, 2018. 46(sup3): p. S264-S273. Lu, Y., et al., Cationic micelle-based siRNA delivery for efficient colon cancer gene therapy. Nanoscale Res Lett, 2019. 14(1). Xin, X., et al., ROS-Responsive Polymeric Micelles for Triggered Simultaneous Delivery of PLK1 Inhibitor/miR-34a and Effective Synergistic Therapy in Pancreatic Cancer. ACS Appl Mater Interfaces, 2019. 11(16): p. 14647-14659. Kang, L., et al., Nanocarrier-mediated co-delivery of chemotherapeutic drugs and gene agents for cancer treatment. Acta Pharm Sin B, 2015. 5(3): p. 169-175. Keeler, A.M., M.K. ElMallah, and T.R. Flotte, Gene Therapy 2017: Progress and Future Directions. Clin Transl Sci, 2017. 10(4): p. 242-248. Fire, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391: p. 806. Chakraborty, C., et al., Therapeutic miRNA and siRNA: Moving from Bench to Clinic as Next Generation Medicine. Mol Ther Nucleic Acids, 2017. 8: p. 132-143. Dai, X. and C. Tan, Combination of microRNA therapeutics with small-molecule anticancer drugs: Mechanism of action and co-delivery nanocarriers. Adv Drug Deliv Rev, 2015. 81: p. 184-197. Saraswathy, M. and S. Gong, Recent developments in the co-delivery of siRNA and small molecule anticancer drugs for cancer treatment. Materials Today, 2014. 17(6): p. 298-306. Burnett, John C. and John J. Rossi, RNA-Based Therapeutics: Current Progress and Future Prospects. Chem Biol 2012. 19(1): p. 60-71. Vicentini, F.T., et al., Delivery systems and local administration routes for therapeutic siRNA. Pharm Res, 2013. 30(4): p. 915-31. Zhou, Y., C. Zhang, and W. Liang, Development of RNAi technology for targeted therapy — A track of siRNA based agents to RNAi therapeutics. J Control Release, 2014. 193: p. 270-281. Suter, S.R., et al., Controlling miRNA-like off-target effects of an siRNA with nucleobase modifications. Org Biomol Chem, 2017. 15(47): p. 10029-10036. Suter, S.R., et al., Structure-Guided Control of siRNA Off-Target Effects. J Am Chem Soc, 2016. 138(28): p. 8667-8669. Sandiford, O.A., et al., Human Aging and Cancer: Role of miRNA in Tumor Microenvironment. Adv Exp Med Biol, 2018. 1056: p. 137-152. Palmini, G., F. Marini, and M. Brandi, What Is New in the miRNA World Regarding Osteosarcoma and Chondrosarcoma? Molecules, 2017. 22(3): p. 417. Wang, F.R., et al., MiR-485-5p inhibits metastasis and proliferation of osteosarcoma by targeting CX3CL1. Eur Rev Med Pharmacol Sci, 2018. 22(21): p. 7197-7204. Zhu, X., L. Chen, and J. Lin, miR-219a-5p represses migration and invasion of osteosarcoma cells via targeting EYA2. Artif Cells Nanomed Biotechnol, 2018. 46(sup3): p. S1004-S1010. Zheng, X.M., C.W. Xu, and F. Wang, MiR-33b inhibits osteosarcoma cell proliferation through suppression of glycolysis by targeting Lactate Dehydrogenase A (LDHA). Cell Mol Biol (Noisy-le-grand), 2018. 64(11): p. 31-35.
Zhang, H., et al., miR-422a inhibits osteosarcoma proliferation by targeting BCL2L2 and KRAS. Biosci Rep, 2018. 38(2). Yang, X., et al., MiR-183 inhibits osteosarcoma cell growth and invasion by regulating LRP6-Wnt/beta-catenin signaling pathway. Biochem Biophys Res Commun, 2018. 496(4): p. 1197-1203. Xu, Y., et al., miR-365 functions as a tumor suppressor by directly targeting CYR61 in osteosarcoma. Biomed Pharmacother, 2018. 98: p. 531-537. Xiao, T., et al., MiR-125b suppresses the carcinogenesis of osteosarcoma cells via the MAPK-STAT3 pathway. J Cell Biochem, 2018. Wang, X., et al., miR-423-5p Inhibits Osteosarcoma Proliferation and Invasion Through Directly Targeting STMN1. Cell Physiol Biochem, 2018. 50(6): p. 2249-2259. Wang, W.T., et al., miR-590-3p is a novel microRNA which suppresses osteosarcoma progression by targeting SOX9. Biomed Pharmacother, 2018. 107: p. 1763-1769. Wang, W., et al., MiR-216b inhibits osteosarcoma cell proliferation, migration, and invasion by targeting Forkhead Box M1. J Cell Biochem, 2018. Wang, N., et al., miR-141-3p suppresses proliferation and promotes apoptosis by targeting GLI2 in osteosarcoma cells. Oncol Rep, 2018. 39(2): p. 747-754. Wang, J., et al., miR-141-3p is a key negative regulator of the EGFR pathway in osteosarcoma. Onco Targets Ther, 2018. 11: p. 4461-4478. Sun, X., et al., miR-143-3p inhibits the proliferation, migration and invasion in osteosarcoma by targeting FOSL2. Sci Rep, 2018. 8(1): p. 606. Wang, D.Z., et al., MiR-218 promotes apoptosis of U2OS osteosarcoma cells through targeting BIRC5. Eur Rev Med Pharmacol Sci, 2018. 22(20): p. 6650-6657. Tao, J., et al., MiR-30a-5p inhibits osteosarcoma cell proliferation and migration by targeting FOXD1. Biochem Biophys Res Commun, 2018. 503(2): p. 1092-1097. Ren, Y.F., et al., miR-144 suppresses proliferation and induces apoptosis of osteosarcoma cells via direct regulation of mTOR expression. Oncol Lett, 2018. 15(1): p. 1163-1169. Miao, J., et al., miR-194 Suppresses Proliferation and Migration and Promotes Apoptosis of Osteosarcoma Cells by Targeting CDH2. Cell Physiol Biochem, 2018. 45(5): p. 1966-1974. Lu, C., et al., miR-18a-5p promotes cell invasion and migration of osteosarcoma by directly targeting IRF2. Oncol Lett, 2018. 16(3): p. 3150-3156. Gao, Y.F., et al., miR-142 suppresses proliferation and induces apoptosis of osteosarcoma cells by upregulating Rb. Oncol Lett, 2018. 16(1): p. 733-740. Chen, T., et al., miR-491-5p inhibits osteosarcoma cell proliferation by targeting PKM2. Oncol Lett, 2018. 16(5): p. 6472-6478. Li, C., et al., miR-202-5p inhibits the migration and invasion of osteosarcoma cells by targeting ROCK1. Oncol Lett, 2018. 16(1): p. 829-834. Pang, P.C., et al., miR-497 as a potential serum biomarker for the diagnosis and prognosis of osteosarcoma. Eur Rev Med Pharmacol Sci, 2016. 20(18): p. 3765-3769. Ren, X., et al., miR-21 predicts poor prognosis in patients with osteosarcoma. Br J Biomed Sci, 2016. 73(4): p. 158-162. Cong, C., et al., Identification of serum miR-124 as a biomarker for diagnosis and prognosis in osteosarcoma. Cancer Biomark, 2018. 21(2): p. 449-454. Gao, F. and F. Xu, Reduced expression of miR-564 is associated with worse prognosis in patients with osteosarcoma. Eur Rev Med Pharmacol Sci, 2018. 22(18): p. 5851-5856. Cai, H., M. Miao, and Z. Wang, miR-214-3p promotes the proliferation, migration and invasion of osteosarcoma cells by targeting CADM1. Oncol Lett, 2018. 16(2): p. 26202628. Liu, X., et al., miR-210 promotes human osteosarcoma cell migration and invasion by targeting FGFRL1. Oncol Lett, 2018. 16(2): p. 2229-2236.
Peng, N., et al., MiR-378 promotes the cell proliferation of osteosarcoma through down-regulating the expression of Kruppel-like factor 9. Biochem Cell Biol, 2018. 96(5): p. 515-521. Bi, Y., Y. Jing, and Y. Cao, Overexpression of miR-100 inhibits growth of osteosarcoma through FGFR3. Tumour Biol, 2015. 36(11): p. 8405-11. Wang, L., et al., miR-205 suppresses the proliferative and migratory capacity of human osteosarcoma Mg-63 cells by targeting VEGFA. Onco Targets Ther, 2015. 8: p. 2635-42. Wei, R., Z. Deng, and J. Su, miR-217 targeting Wnt5a in osteosarcoma functions as a potential tumor suppressor. Biomed Pharmacother, 2015. 72: p. 158-64. Yang, D., G. Liu, and K. Wang, miR-203 Acts as a Tumor Suppressor Gene in Osteosarcoma by Regulating RAB22A. PLoS One, 2015. 10(9): p. e0132225. Xu, E., et al., miR-146b-5p promotes invasion and metastasis contributing to chemoresistance in osteosarcoma by targeting zinc and ring finger 3. Oncol Rep, 2016. 35(1): p. 275-83. Pu, Y., et al., The miR-34a-5p promotes the multi-chemoresistance of osteosarcoma via repression of the AGTR1 gene. BMC Cancer, 2017. 17(1): p. 45. Pu, Y., et al., MiR-34a-5p promotes multi-chemoresistance of osteosarcoma through down-regulation of the DLL1 gene. Sci Rep, 2017. 7: p. 44218. Zou, Y., et al., miR-133b induces chemoresistance of osteosarcoma cells to cisplatin treatment by promoting cell death, migration and invasion. Oncol Lett, 2018. 15(1): p. 1097-1102. Xu, M., et al., MiR-34c inhibits osteosarcoma metastasis and chemoresistance. Med Oncol, 2014. 31(6): p. 972. Wang, S.N., et al., miR-491 Inhibits Osteosarcoma Lung Metastasis and Chemoresistance by Targeting alphaB-crystallin. Mol Ther, 2017. 25(9): p. 2140-2149. Yan, H., et al., miR-340 alleviates chemoresistance of osteosarcoma cells by targeting ZEB1. Anticancer Drugs, 2018. 29(5): p. 440-448. Long, X. and X.-J. Lin, P65-mediated miR-590 inhibition modulates the chemoresistance of osteosarcoma to doxorubicin through targeting wild-type p53-induced phosphatase 1. J Cell Biochem, 2019. 120(4): p. 5652-5665. Kundu, A.K., et al., Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma. PLoS ONE, 2017. 12(6): p. e0179168. Liu, M., D. Wang, and N. Li, Che-1 gene silencing induces osteosarcoma cell apoptosis by inhibiting mutant p53 expression. Biochem Biophys Res Commun, 2016. 473(1): p. 168-173. Pereira, P., et al., Smart micelleplexes as a new therapeutic approach for RNA delivery. Expert Opin Drug Deliv, 2017. 14(3): p. 353-371. Zhang, Y., et al., Charged group surface accessibility determines micelleplexes formation and cellular interaction. Nanoscale, 2015. 7(17): p. 7559-64. Jones, C.H., et al., Overcoming nonviral gene delivery barriers: perspective and future. Mol Pharm, 2013. 10(11): p. 4082-4098. Yu, H., et al., Overcoming Endosomal Barrier by Amphotericin B-Loaded Dual pHResponsive PDMA-b-PDPA Micelleplexes for siRNA Delivery. ACS Nano, 2011. 5(11): p. 9246-9255. Yu, H., et al., Reversal of lung cancer multidrug resistance by pH-responsive micelleplexes mediating co-delivery of siRNA and paclitaxel. Macromol Biosci, 2014. 14(1): p. 100-9. Sun, T.M., et al., Simultaneous delivery of siRNA and paclitaxel via a "two-in-one" micelleplex promotes synergistic tumor suppression. ACS Nano, 2011. 5(2): p. 1483-94. Wang, D., et al., Acid-Activatable Versatile Micelleplexes for PD-L1 Blockade-Enhanced Cancer Photodynamic Immunotherapy. Nano Lett, 2016. 16(9): p. 5503-13.
Yu, H., et al., Triple-Layered pH-Responsive Micelleplexes Loaded with siRNA and Cisplatin Prodrug for NF-Kappa B Targeted Treatment of Metastatic Breast Cancer. Theranostics, 2016. 6(1): p. 14-27. Chen, M., et al., Synergistic antitumor efficacy of redox and pH dually responsive micelleplexes for co-delivery of camptothecin and genes. Acta Biomater, 2017. 49: p. 444-455. Lehner, R., et al., Efficient Receptor Mediated siRNA Delivery in Vitro by Folic Acid Targeted Pentablock Copolymer-Based Micelleplexes. Biomacromolecules, 2017. 18(8): p. 2654-2662. Magalhaes, M., et al., miR-145-loaded micelleplexes as a novel therapeutic strategy to inhibit proliferation and migration of osteosarcoma cells. Eur J Pharm Sci, 2018. 123: p. 28-42. Shen, S., et al., Co-delivery of platinum drug and siNotch1 with micelleplex for enhanced hepatocellular carcinoma therapy. Biomaterials, 2015. 70: p. 71-83. Lee, S.Y., et al., A theranostic micelleplex co-delivering SN-38 and VEGF siRNA for colorectal cancer therapy. Biomaterials, 2016. 86: p. 92-105. Schlacher, K., A new road to cancer-drug resistance. Nature, 2018. 563(7732): p. 478480. He, C., et al., Core-shell nanoscale coordination polymers combine chemotherapy and photodynamic therapy to potentiate checkpoint blockade cancer immunotherapy. Nat Commun, 2016. 7: p. 12499. Xu, J., et al., Near-Infrared-Triggered Photodynamic Therapy with Multitasking Upconversion Nanoparticles in Combination with Checkpoint Blockade for Immunotherapy of Colorectal Cancer. ACS Nano, 2017. 11(5): p. 4463-4474. Yang, G., et al., Smart Nanoreactors for pH-Responsive Tumor Homing, MitochondriaTargeting, and Enhanced Photodynamic-Immunotherapy of Cancer. Nano Lett, 2018. 18(4): p. 2475-2484. Li, E., et al., MiR-145 inhibits osteosarcoma cells proliferation and invasion by targeting ROCK1. Tumour Biol, 2014. 35(8): p. 7645-50. Wu, P., et al., miR-145 promotes osteosarcoma growth by reducing expression of the transcription factor friend leukemia virus integration 1. Oncotarget, 2016. 7(27): p. 42241-42251. Nam, J.P., S. Kim, and S.W. Kim, Design of PEI-conjugated bio-reducible polymer for efficient gene delivery. Int J Pharm, 2018. 545(1-2): p. 295-305. Ryu, N., et al., Effective PEI-mediated delivery of CRISPR-Cas9 complex for targeted gene therapy. Nanomedicine, 2018. 14(7): p. 2095-2102. Tong, W.Y., et al., Delivery of siRNA in vitro and in vivo using PEI-capped porous silicon nanoparticles to silence MRP1 and inhibit proliferation in glioblastoma. J Nanobiotechnol, 2018. 16(1): p. 38. Sun, K., et al., Dextran-g-PEI nanoparticles as a carrier for co-delivery of adriamycin and plasmid into osteosarcoma cells. Int J Biol Macromol, 2011. 49(2): p. 173-80. Hsu, H.K., et al., Development and In Vitro Evaluation of Linear PEI-Shelled Heparin/Berberine Nanoparticles in Human Osteosarcoma U-2 OS Cells. Molecules, 2018. 23(12). Zhou, Y., et al., Cyclam-Modified PEI for Combined VEGF siRNA Silencing and CXCR4 Inhibition To Treat Metastatic Breast Cancer. Biomacromolecules, 2018. 19(2): p. 392401. Merkel, O.M. and T. Kissel, Quo vadis polyplex? J Control Release, 2014. 190: p. 415423. He, H., et al., Reversibly Cross-Linked Polyplexes Enable Cancer-Targeted Gene Delivery via Self-Promoted DNA Release and Self-Diminished Toxicity. Biomacromolecules, 2015. 16(4): p. 1390-1400.
Saeg, F. and M. Anbalagan, Breast cancer stem cells and the challenges of eradication: a review of novel therapies. Stem Cell Investig, 2018. 5: p. 39. Vengoji, R., et al., Novel therapies hijack the blood–brain barrier to eradicate glioblastoma cancer stem cells. Carcinogenesis, 2018. 40(1): p. 2-14. Brown, H.K., M. Tellez-Gabriel, and D. Heymann, Cancer stem cells in osteosarcoma. Cancer Lett, 2017. 386: p. 189-195. Lee, S., et al., Spotlight on nano-theranostics in South Korea: applications in diagnostics and treatment of diseases. Int J Nanomedicine, 2015. 10 Spec Iss: p. 3-8. Sneider, A., et al., Remotely Triggered Nano-Theranostics For Cancer Applications. Nanotheranostics, 2017. 1(1): p. 1-22. Zhang, S., et al., Proteomic investigation of resistance to chemotherapy drugs in osteosarcoma. Technol Health Care, 2018. 26(1): p. 145-153. Niveditha, D., et al., A global transcriptomic pipeline decoding core network of genes involved in stages leading to acquisition of drug-resistance to cisplatin in osteosarcoma cells. Bioinformatics, 2018. 35(10): p. 1701-1711. Kazemi Oskuee, R., et al., Investigating the influence of polyplex size on toxicity properties of polyethylenimine mediated gene delivery. Life Sci, 2018. 197: p. 101-108. Monnery, B.D., et al., Cytotoxicity of polycations: Relationship of molecular weight and the hydrolytic theory of the mechanism of toxicity. Int J Pharm 2017. 521(1): p. 249258. Alvarez-Lorenzo, C. and A. Concheiro, Bioinspired drug delivery systems. Curr Opin Biotechnol, 2013. 24(6): p. 1167-1173. Zhai, Y., et al., Preparation and Application of Cell Membrane-Camouflaged Nanoparticles for Cancer Therapy. Theranostics, 2017. 7(10): p. 2575-2592. Zhen, X., P. Cheng, and K. Pu, Recent Advances in Cell Membrane-Camouflaged Nanoparticles for Cancer Phototherapy. Small, 2018: p. e1804105. Wang, D., et al., Erythrocyte–Cancer Hybrid Membrane Camouflaged Hollow Copper Sulfide Nanoparticles for Prolonged Circulation Life and Homotypic-Targeting Photothermal/Chemotherapy of Melanoma. ACS Nano, 2018. 12(6): p. 5241-5252. Crenn, V., et al., Bone microenvironment has an influence on the histological response of osteosarcoma to chemotherapy: retrospective analysis and preclinical modeling. Am J Cancer Res, 2017. 7(11): p. 2333-2349. Sharma, A., C. Cinti, and E. Capobianco, Multitype Network-Guided Target Controllability in Phenotypically Characterized Osteosarcoma: Role of Tumor Microenvironment. Front Immunol, 2017. 8: p. 918. Zheng, Y., et al., Mesenchymal stem cells in the osteosarcoma microenvironment: their biological properties, influence on tumor growth, and therapeutic implications. Stem Cell Res Ther, 2018. 9(1): p. 22. Kawano, M., et al., Interaction between human osteosarcoma and mesenchymal stem cells via an interleukin-8 signaling loop in the tumor microenvironment. Cell Commun Signal, 2018. 16(1): p. 13. Lan, M., et al., Extracellular vesicles-mediated signaling in the osteosarcoma microenvironment: Roles and potential therapeutic targets. J Bone Oncol, 2018. 12: p. 101-104. Xu, J.F., et al., Exosomes containing differential expression of microRNA and mRNA in osteosarcoma that can predict response to chemotherapy. Oncotarget, 2017. 8(44): p. 75968-75978. Brady, J.V., et al., A Preliminary Proteomic Investigation of Circulating Exosomes and Discovery of Biomarkers Associated with the Progression of Osteosarcoma in a Clinical Model of Spontaneous Disease. Transl Oncol, 2018. 11(5): p. 1137-1146.
237.
Wang, J.W., et al., Exosomal miR-1228 from cancer-associated fibroblasts promotes cell migration and invasion of osteosarcoma by directly targeting SCAI. Oncol Res, 2018.
Table 1: Different approaches regarding nanoparticles formulation for OS therapy.
Nanoparticle
Nanoparticle Composition
Loading
Findings
ref
Liposomes
Egg phosphatidylcholine; derivatized
Doxorubicin
EE 84%; D 96 nm; <37% release
[51]
distearyl phosphatidylethanolamine
(t=48h); increased toxicity 1.8-4.6
(mPEG2000- DSPE); cholesterol
times (OS cells);
ZnO
Apoptosis and autophagy ZnO
Nanoparticle
NPs-mediated cell death (OS cells).
Dextran-based
Lipid-modified dextran,
microRNA-
Delivering of siRNAs to OS cancer
Nanoparticles
thiolated-dextran derivative,
199a-3p
cells; S 351.6±2.5 nm; ZP -
PEGylated-dextran derivative
[52]
[53]
25.3±7.47 mV
PLGA
poly(lactic-co-glycolic acid) +
Paclitaxel +
EE: 92.5 ± 5.6% DL: 13.6 ± 2.8%
nanoparticles
PEGylated poly(lactic-co-glycolic
etoposide
S: 100 ± 3.68 nm; 15 ± 3.5% PCT
acid)
[54]
released, 25 ± 2.6% ETP released (t=24h)
Lipid-coated
1,2-dilauroyl-sn-glycero-3-phos-
Curcumin +
S: 100nm; ZP: -33,4mV;
polymeric
phocholine (DLPC), cholesterol
doxorubicin
EE(DOX):85%; EE(CUR):80%;
nanoparticle
(CHOL), PEGylated distearyl
55% (DOX) released, 50% (CUR)
phosphatidylethanolamine (DSPE),
released (t=12h).
[55]
Poly (lactic-co-glycolic acid) Polymeric
poly(ethylene glycol)-poly[2-
Zinc
100-fold higher cytotoxicity when
micelles
(methylacryloyl)ethylnicotinate]
phthalocyanine
compared to free ZnPc; EE: 89,4%;
(PEG-PMAN), nicotinate
(ZnPc)
DL:8,2%; D=30 nm; 25% ZnPc
[56]
release (t=14 days). Coated gold
Polyacrilic acid-coated gold nanorods
Photothermal
Length:44,3nm; Width:10,8 nm;
nanorods
(PAA-GNR)
therapy
enhanced thermal conductivity;
[57]
superior biocompatibility; higher efficiency of photothermic therapy. Hydroxyapatite
Highlights Osteosarcoma represents the main cancer affecting bone tissue New strategies are imperatively needed for osteosarcoma targeting therapies Gene therapy has emerged as a promising strategy for cancer treatment Non-viral nanosystems have been developed for efficient nucleic acid delivery Micelleplexes are innovative, tunable and multifunctional nucleic acid carriers
