Nanodelivery Systems Face Challenges and Limitations in Bone Diseases Management

1 Introduction

Bone diseases encompass a variety of skeletal-related disorders, and can be divided into subclasses including genetic, metabolic, degenerative, traumatic, malignant (metastatic and primary), and myeloma-related disorders. Bone disorders and their potential complications have a major impact on an individual's life, causing disability, reduced quality of life, and a downward spiral in physical and mental health that for some ultimately results in death.[1] Bone is considered one of the vital organs of the body because the skeleton provides mechanical support for stature and locomotion, protects vital organs, and controls mineral homeostasis.[2] Moreover, recent studies have highlighted the role of bone as an endocrine organ that modulates its own metabolic functions through the production of hormones by bone cells, controlling mineral ion homeostasis (e.g., through Fibroblast growth factor 23 production) and energy balance (e.g., through osteocalcin production).[3] The integrity of the skeleton is maintained by continuous bone remodeling. However, some bone disorders develop from alterations in this physiological process, which can be caused by factors including hormones, age, physical activity, drugs, and comorbidities.[4]

Bone disorders have a high rate of morbidity and mortality, and the majority still lack a clinical solution.[5] Though changes in treatment have improved patients’ survival, the balance between side effects and treatment efficacy unfortunately remains a battle. Therefore, there is still a concrete need to develop safer and more effective treatments. Many solutions have been explored, including targeted drug delivery systems, which seem to be a potential winning strategy.[6] Nanoparticle (NP)-based approaches combined with the available drugs allow researchers to overcome some of the side effects associated with current therapies.[6]

NPs as drug delivery systems have been extensively applied for many different diseases, from tumors to regenerative/inflammatory pathologies.[7, 8] NPs have proven a favorable platform for specific tissue targeting, since they can develop stable interactions with different ligands, increase drug loading efficiency of both hydrophilic and hydrophobic molecules, and control delivery of micro- and macromolecules. In particular, NPs assure (i) increased drug solubility; (ii) prolonged drug stability as a result of the protective shield that reduces drug metabolism after administration and avoids fast clearance by filtering organs; (iii) enhanced transport ability across cell membranes; (iv) reduction of drug resistance mediated by extrusion pumps; (v) specific delivery of therapeutics to targeted tissue; (vi) controlled release of therapeutic cargo; (vii) reduced systemic adverse effects on healthy tissues or organs; and (viii) multimodal therapies.[9] Due to these properties, nanomedicine has brought forth numerous successful formulations that have reached different stages of clinical trials or approval by the U.S. Food and Drug Administration (FDA), including the recent successful vaccine formulations against SARS-COV-2 by Pfizer and Moderna.[10]

In this review, we summarize the progress made in the development of NPs for the treatment of bone disorders, as well as highlight possible strategies to overcome current challenges, as well as potential uses of NPs for malignancies and bone disorders. Finally, we will discuss potential limitations in the translation of such technologies.

1.1 Bone Architecture and Function

Bone, despite its apparent rigid structure, is a remarkably complex and dynamic tissue. Its structure is maintained by the bone remodeling process, in which old or damaged bone is removed by osteoclasts (through bone resorption) and replaced with newly formed bone by osteoblasts (through bone formation) with no change in bone mass or quality (Figure 1).[11, 12] This plastic homeostasis allows the skeleton to change in size during childhood and provides the bone the unique ability to repair micro- and macro-damages, such as fractures.[13, 14]

image Bone remodeling involves the removal of mineralized bone by osteoclast (resorption) followed by the formation of bone matrix by osteoblast (deposition) and subsequent mineralization. The process of bone remodeling maintains bone structure to meet changing mechanical needs and repairs damage in bone matrix. Figure 1 was created using Biorender.com.

The structure of bone can be divided into the inner trabecular bone and outer cortical bone. Trabecular bone, the “core” of skeletal structure, is very porous and houses the bone marrow, while encompassing only 20% of the total bone mass. Conversely, cortical bone is dense and organized into units called osteons, with lower blood circulation (Figure 2A).[15] Eighty percent of bone mass is composed of a mixture of calcium phosphate salts (hydroxyapatite [HA]) and collagen, making bones a highly mineralized organ.[16] This structure makes bones exceptionally resistant to mechanical stress and provides the strength to structurally support the rest of the body (Figure 2B).[17]

image Schematic representation of bone structure. Reproduced with permission.[18] Copyright 2018, Elsevier B.V. 1.2 Challenges in Bone Treatments

Bones are highly vascularized, but the structure of the vascular network can vary greatly depending on the skeletal site, age,[19] and presence of pathologic conditions,[20] affecting the diffusion of the drug from the bloodstream to the target site.[21] Interestingly, the absence of a basal membrane in some bone blood vessels makes them especially permeable to large molecules. This physiological feature can favor NP accumulation within the targeted bone, similar to the enhanced permeability and retention (EPR) effect present in many tumors (Figure 3).[22]

image Schematic representation of IV administered NPs path from the blood flow through the bone tissue: A) NPs extravasate from the bone capillaries either through transcellular pathways such as receptor-mediate transcytosis or through a paracellular pathway, using capillary fenestrae of varying size; B) the NPs reach the bone marrow and can interact with its ECM, or with the resident immune and hematopoietic cells populations; C) From the bone marrow, NPs can diffuse in the trabecular bone, encountering different bone-specific cells populations and its denser mineralized ECM. Figure 3 was created using Biorender.com.

On the other hand, the mineralized nature of the bone extracellular matrix poses an exceptional barrier to NP diffusion from blood vessels into the surrounding tissue. In particular, the small gaps between mineralized bone fibers (lamellae) are only a few hundred nanometers large, imposing a threshold (100–300 nm) for the maximum size of NPs targeting the bone.[23, 22] However, even for NPs ranging between 100 and 200 nm or below, the limited space makes tissue penetration challenging (Figure 3).

From a clinical and experimental point of view, differences in bone mineral density, mechanical cues, and biochemical composition between different species (human, dog, sheep, mouse, and rat) need to be considered when choosing an appropriate animal model for bone research. This makes the application of NPs for bone-related diseases far from trivial.[24]

The majority of the NP-based treatment studies reported in the literature are focused on the use of NPs for cancer treatment, and different strategies have been investigated for primary bone tumors and bone metastases.

2 Bone Tumor Diseases 2.1 Bone as the Primary Site of Malignances

Osteosarcoma (OS), chondrosarcoma (CS), and Ewing sarcoma (ES) are the three most common primary bone tumors, accounting for 70% of all cases.[25] They are considered rare tumors and comprise a diverse group of malignant neoplasms.[25] Bone sarcomas are of mesenchymal origin; mesenchymal stem cells (MSCs) can be either the progenitor of tumor cells,[26, 27] or stromal cells that participate in tumor development and progression.[28, 29] These tumors can originate in various locations within the bone structure, anywhere from the medullary cavity to the periosteum.[30]

While progress has been made toward improving patient outcomes, the survival rate remains below 30% for patients with metastatic disease.[31] The difficulties in treating bone tumors are primarily due to the high biological and anatomical heterogeneity of the cases as well as the complication presented by the high level of metastasis.[32]

2.1.1 Osteosarcoma

OS is the most common pediatric bone cancer.[33, 34] Approximately 1200 patients are diagnosed with OS annually in the United States, making OS the third most common childhood malignancy, with incidence at a median age of 12 years for girls and 16 years for boys.[35] OS classically develops in the metaphysis of long bones, such as distal femurs and proximal tibiae.[36] This tumor usually arises from malignant primitive MSCs that differentiate into osteoblasts under physiological conditions, but produce a malignant osteoid matrix during progression of malignancies.[37]

OS is known to have a significantly poorer prognosis compared to most other pediatric cancers.[38] The primary reason for treatment failure and recurrence in OS is the high incidence of distant metastatic lesions, which in 80% of cases spread to the lungs.[39]

OS metastasis mainly develops due to low responsiveness to chemotherapy, uncontrolled proliferation of cancer stem cells (CSCs), and drug resistance.[30] Therefore, CSC population represents one of the most important and difficult targets for improvement of the long-term efficacy of chemotherapeutics.[40]

2.1.2 Ewing Sarcoma

ES is the second most common primary bone tumor after OS, representing 10–15% percent of all cases.[41, 42] Like OS, the diagnosis is primarily made among pediatric patients. One of the unique features of this malignancy is that it can originate in either the bone or in soft tissues.[43] The cellular origin of ES remains unclear, and is still a matter of debate. However, recent studies have demonstrated that MSCs are also the most likely cells of origin for this sarcoma.[44]

2.1.3 Chondrosarcoma

CS is the third most common primary tumor of the musculoskeletal system; although improperly classified as a bone tumor, it can be included in this class because of its similarity to osteosarcomas and proximity to the bone tissue.[45] CS is considered a family of different diseases, including primary and secondary CS. It also includes more rare diseases such as dedifferentiated CS, mesenchymal CS, and clear-cell CS.[46]

Though all unique, CS diseases share the characteristic of abnormal deposition of cartilage tissue. Due to the increased deposition of extracellular matrix that form a physical barrier against treatments, systemic chemotherapy is generally not considered the most effective therapeutic approach. However, there have been some recent successful attempts at developing NPs for CS treatment, which represent a new frontier, and are the reason we have included it in this review.

2.2 Bone as the Secondary Site of Malignancies

The formation of metastases is a complex, multistep process in which malignant tumor cells spread from their primary tumor lesion to distant organs.[47] Tumors most often metastasize to the bone, because its microenvironment helps tumor cells to adhere and proliferate due to the release of survival and growth promoting factors during the process of bone formation and resorption.[48]

Most bone metastases (BMs) originate from renal cell carcinoma, prostate cancer, breast cancer, and lung cancer, and their incidence usually depends on the tumor stage at diagnosis.[48] Metastatic spread accounts for ≈90% of all cancer-related deaths in solid malignancies,[49] and there has been little improvement in the five-year survival rate over the past decade.[49] Therefore, new research needs to be conducted on novels ways to treat BMs.

3 Current Treatments for Primary Bone Tumors and Established Bone Metastases

Current treatments for primary bone tumors vary depending on the type and grade of the malignancy.[40, 50, 51] OS and ES tend to be more chemo-sensitive and are primarily treated with neoadjuvant chemotherapy prior to wide surgical resection.[40] CS, on the other hand, is generally resistant to both chemotherapy and radiation; thus, the primary treatment is generally wide surgical excision at the time of diagnosis.[52] The exception to this is grade 1 CS within the extremities, which is treated with intra-lesion curettage alone due to the low rate of distant metastasis.[53] The surgical resection method varies depending on the location and size of the tumor and includes amputation, en bloc excision, and limb salvage techniques.[54]

Except for grade 1 CS, surgical resection alone is often not curative due to the development of micro-metastases that can result in tumor recurrence.[55] Therefore, surgical resection is often used as a method of palliative treatment to alleviate pain from the tumor mass.[56]

Neoadjuvant chemotherapy is used to reduce the tumor mass and number of BMs prior to surgical intervention, while adjuvant chemotherapy is used after surgery to reduce the chance of tumor recurrence.[57, 58] However, though chemotherapy is one of the first-line treatment options for patients with systemic disease, it is also associated with severe adverse effects including fever, neutropenia, hypersensitivity reactions, and cardiotoxicity due to the non-specific drug biodistribution.[59] Moreover, chemotherapy efficacy is also impaired by the bone tissue dense extracellular matrix and by the bone marrow microenvironment which provide a protective niche for cancer cells increasing the development of treatment resistance.[60] The most common chemotherapeutics used individually or in combination for primary and metastatic bone tumors are doxorubicin, cisplatin, methotrexate (MTX), cyclophosphamide, and ifosfamide.[61] In particular, patients diagnosed with primary and metastatic OS are treated with drug combination therapy (high-dose cycles of doxorubicin, cisplatin, and MTX).[62-64] Radiotherapy and radiopharmaceuticals can be used alone or in combination with surgery or chemotherapy. External-beam radiotherapy is used to kill cancer cells, reduce bone pain, and decrease fractures.[65] The use of radiopharmaceuticals as well as other radiation-enhancing materials in combination with traditional radiotherapy has attracted much attention in the last years thanks to their ability to control the amount of administered radiation, reducing the notorious adverse effects.[66] Furthermore, radiopharmaceuticals can be used as theranostic tools to perform both tumor imaging and treatment in a single step.[66]

For rare bone malignancies of ES, the most commonly applied therapeutic regimen, known as VDC/IE, is based on a first therapy cycle with vincristine, doxorubicin, and cyclophosphamide, and a second cycle of ifosfamide and etoposide.[67] This therapy is normally followed by either radiotherapy or surgery.[68, 69] Similarly, for the treatment of CS, vincristine, doxorubicin, cyclophosphamide, and etoposide are often used.[70] In the case of metastatic CS, vincristine, doxorubicin, and cyclophosphamide are preferred.[70, 71]

Recently, bisphosphonates (BPs) have been used more frequently in the treatment of bone primary tumors and BMs.[72, 73] They have been used to increase bone targeting thanks to their ability to enhance binding affinity of calcium ions on HA.[60]Three generations of BPs have been approved in the clinic for bone regeneration and bone metastases treatment. Therefore, BPs have been commonly prescribed for prevention and treatment of a variety of bone conditions in which an imbalance between bone formation and bone resorption underlies disease pathology.[60, 71-73]

Overall, dramatic improvements in treatment regimens have made it possible for most patients with bone tumors in the extremities to avoid full limb amputation, allowing prosthetics or limb salvage surgery use instead to attempt to restore limb function.[74]

4 Nanoparticle Treatments for Primary Bone Tumors 4.1 Osteosarcoma 4.1.1 Nanoparticles and Traditional Chemotherapeutics

NPs have been used to improve the efficacy of well-established drugs already used in the treatment of primary bone tumors (Table 1, Figure 4). MTX is a common chemotherapeutic whose mechanism of action involves impairing DNA bases to affect DNA synthesis.[75] It has been demonstrated to exert good efficacy against OS in the clinical setting but unfortunately is associated with severe side effects, especially in the pediatric population, due to its medullary suppressive activity.[76] To be effective, MTX must be used in very high doses, as well as in combination with other chemotherapeutic drugs.[74, 76] To improve MTX pharmacokinetics, Ray et al.[77, 78] delivered MTX loaded into PLGA coated Mg-Al-layered double hydroxide NPs in a subcutaneous OS murine model. Compared to an equivalent amount of free MTX, MTX-loaded NPs showed more efficient accumulation within the tumor, significantly increased MTX clearance time (≈fivefold), and longer retention of MTX (fivefold higher half-life), resulting in increased MTX efficacy. In addition, systemic and organ toxicity was reduced compared to free MTX. Finally, these NPs significantly decreased the tumor growth rate, resulting in tumors that were eight times smaller, and prolonging survival time.

Table 1. Nanoparticles treatment for primary bone tumors Study Delivered agent(s) NP Drug class Study design Results Osteosarcoma Ray et al.[74] Methotrexate PLGA coated Mg-Al-layered double hydroxide Antimetabolite In vivo (murine model) Increased effect of methotrexate with higher accumulation in the tumor. Reduced systemic toxicity compared to free methotrexate Martinez-Carmona et al.[77] Doxorubicin Mesoporous silica Topoisomerase inhibitor In vitro Selective cell toxicity to OS tumor cells compared to healthy pre-osteoblast cells Kallus et al.[80] Ponatinib, nintedanib, PD173074 Liposomal formulations Tyrosine kinase inhibitor In vitro/in vivo (murine model) Anti-tumor effect similar to the free drug on OS cell lines Inhibition of downstream target receptors signaling In vivo tumor growth reduction of 60% compared to free PON Zinger et al.[81] Ponatinib Leukosomes Tyrosine kinase inhibitor In vitro Similar or better cytotoxic effect in comparison with the free Ponatinib Enhanced effect in reducing the phosphorylation of ponatinib molecular targets compared to free ponatinib Wang et al.[89] Selenite HA Cytotoxic agent In vivo (murine model) In vivo, reduced tumor growth of 50% Reduction of lung metastases as well as improving healthy organ functions in vivo compared to empty hydroxyapatite NPs Chen et al.[93] Salinomycin Lipid polymer Diterpene glycoside In vitro/in vivo (murine model) Significantly improved antineoplastic activity toward OS and CSC cells by inhibiting EGFR+ and CD133+ both in 2d and 3d Reduced tumor growth in vivo Gui et al.[95] All-trans-retinoic acid Lipid polymer Retinoid In vitro/in vivo (murine model) Specific cytotoxicity against CD133+ OS cells both in 2d and 3d Suppressed tumor growth in vivo Wang et al.[98] TRAIL coded plasmids PAMAM dendrimers N/A In vitro/in vivo (murine model) Cytotoxicity against human OS cells in vitro Reduced tumor growth in vivo with no toxic effects Tuohy et al.[101] SPION Cationic liposomes N/A In vivo (murine model) Achieved immunogenic cell death via thermal ablation resulting in immune antitumor response Yu et al.[105] Zn–Ph–BSA Complex PEG-PMAN block polymer N/A In vitro/in vivo (murine model) Extended blood circulation time with preferential accumulation in OS cells Substantially increased apoptosis and autophagy of OS cells both in vitro and in vivo Martella et al.[106] Paclitaxel Multi-modal keratin NP with Chlorin-e6 Antimicrotubule agent In vitro/ in vivo (murine model) Substantial reduction in OS cell viability in vitro Lenna et al.[107] Photosensitizer tetrasulfonated aluminum phthalocyanine Multimodal MSC fluorescent NP Photodynamic agent In vitro MSCs were able to kill OS cells and decrease tumor growth by 68% after two cycles of photoactivation compared to both control groups (PBS and free AlPcS4) Prasad et al.[109] MTX, BSA HA Anti-metabolite In vitro Demonstrated a synergistic cytotoxic effect on OS cell with this combinational regimen versus individual free drugs Yan et al.[93] Saporin Polyboronated dendrimer with poly-(α, β)-DL aspartic acid coating Ribosome inactivating protein (RIP) In vitro/ in vivo (murine model) Enhanced accumulation into the bone Inhibited tumor growth and reduced tumor size Ewing sarcoma Jordan et al. [112] SN-38 Gold Irotecan metabolite In vivo (murine model) Demonstrates selective toxicity against ES cells expressing EWS-FLI1 mRNA with substantially reduced ES tumor growth in vivo Chondrosarcoma Trucco et al.[114] Doxorubicin temsirolimus Liposomal formulations Topoisomerase inhibitor Clinical, phase II RCT The therapy is well-tolerated by patients with a 53% 60-day response rate NP = nanoparticles; OS = osteosarcoma; PLGA = poly(lactic-co-glycolic acid); DNA = deoxyribonucleic acid; EGFR = epidermal growth factor receptor; TRAIL = tumor necrosis factor-related apoptosis-inducing ligand; SPION = superparamagnetic magnetite nanoparticles; HA = hydroxyapatite; MSC = mesenchymal stem cells; AMF = alternating magnetic field; Zn–Ph = zinc–phthalocyanine; BSA = bovine serum albumin; PEG = poly(ethylene glycol); PMAN = poly[2-(methylacryloyl)ethyl nicotinate]; TRAIL = tumor necrosis factor-related apoptosis inducing ligand; PAMAM = polyamidoamine; RCT = randomized controlled trial; NR = not recorded

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