Recent advances in drug delivery systems for osteosarcoma therapy and bone regeneration

Abstract

Osteosarcoma, the predominant bone malignancy, poses significant challenges due to its high metastatic potential and recurrence rates. Accounting for a substantial percentage of malignant bone tumors, osteosarcoma particularly affects children and adolescents. Despite standard treatment combining surgery and multi-drug chemotherapy, systemic drug administration presents limitations, leading to compromised patient quality of life and severe side effects. New strategies are needed to address these challenges and enhance efficacy while minimizing toxicity. Here, we explore drug delivery platforms in the context of osteosarcoma treatment. We delve into both systemic and local delivery approaches, highlighting recent advances in controlled drug release triggered by various stimuli, modifications for targeted delivery, and co-delivery of chemotherapeutics using nano-platforms. Additionally, we discuss innovations in local delivery methods, including implantable nanoparticles, injectable hydrogels, and scaffolds. Despite these advancements, challenges and limitations persist, emphasizing the need for continued research. We conclude by offering perspectives on the potential of multifunctional scaffolds in revolutionizing osteosarcoma drug delivery, thereby paving the way for improved patient survival and enhanced quality of life.

Introduction

Osteosarcoma (OS), the most common bone malignancy, arises from bone and cartilage and accounts for approximately 55% of all primary malignant bone tumors¹. OS is the third most common malignancy among children and adolescents between the ages of 12 and 18², following leukemia and lymphoma, with a yearly incidence of 5.6 cases per million in children under the age of 15³. OS is notorious for its high rates of metastasis and recurrence, with 15% to 20% of patients presenting clinically detectable metastases at diagnosis. The lungs, the primary site of metastasis, account for over 85%, while bone represents the second most common distant disease site⁴. Recurrent OS affects 30-50% of those with initial localized disease and 80% of patients with metastatic conditions, with the lungs being the predominant site of recurrence⁵.

Standard OS treatment involves a combination of surgery and multi-drug chemotherapy. Surgical intervention aims to excise the tumor with optimal margins while preserving functionality. Pre- and post-operative chemotherapy, consisting of doxorubicin (DOX), methotrexate (MTX), and cisplatin (CDDP), aims to shrink the tumor pre-resection and eliminate residual tumor post-resection⁶. However, systemic chemotherapy presents challenges such as invasiveness and pain, significantly compromising patients’ quality of life. Systemic drug administration faces limitations in reaching neoplastic cells distant from tumor vessels, leading to suboptimal drug concentration in cancerous bone tissue. This arises from factors like drug instability in blood, protein binding, and liver Kupffer cell clearance, resulting in dose-related toxicity⁷. Consequently, high systemic doses are often necessary, causing severe side effects such as myelosuppression, hepatotoxicity, cardiotoxicity, and fatal central nervous system disturbances⁸. More than 30% of OS patients are either resistant to current chemotherapy or experience life-threatening complications, ultimately leading to the progression of metastases and mortality⁹.

To address these challenges, it is crucial to develop novel strategies with reduced toxicity and enhanced efficacy to improve patient survival and quality of life. Recently, nanoparticle-based drug delivery platforms have been introduced to deliver anti-cancer agents for OS. These nanoparticle systems have shown several advantages over traditional systemic chemotherapy for treating OS, including reduced adverse effects, improved drug pharmacokinetics, and extended circulation time in the bloodstream. Despite these significant advantages, this approach suffers from notable limitations, primarily that only a small fraction of systemically administered drugs, often less than 5%, reach the targeted tumor site. Consequently, recent efforts have shifted towards local drug delivery using multiple approaches. The local delivery strategy bypasses the limitations encountered by nanoparticles (NPs) in the bloodstream before reaching the target site, especially in bone tissue, and offers greater potential for the effective release of anti-cancer drugs to treat OS.

In this comprehensive review, we delve into common NPs used as drug carriers in OS, highlighting recent advances in controlled drug release strategies triggered by various stimuli, modifications for targeted drug delivery, and co-delivery of chemotherapeutics using nano-platforms. Additionally, we explore advancements in local delivery, encompassing implantable NPs, injectable hydrogels, and scaffolds (Fig. 1). The review concludes by outlining the challenges and limitations of multifunctional scaffolds in OS drug delivery, including the impact of therapeutic agents on healthy cells, the limitations of current tumor models, and the need for more relevant preclinical animal models.

Fig. 1: Standard and emerging osteosarcoma treatment strategies.

An illustration of current standard treatment for osteosarcoma alongside new alternative drug delivery methods, providing an outline of the review article’s structure. Created with BioRender.com.

Systemic NPs-based drug delivery

Within the past few decades, the field of nanotechnology and nanomedicine has remarkably developed and actively been applied to various diseases such as cancer, infection, skin-related diseases, cardiovascular-related diseases, and regeneration like bone, teeth, and wound healing¹⁰. The majority of research publications in the field of nanomedicine focus on the use of NPs in drug delivery, mainly as drug carriers to deliver proteins, growth factors, and other chemical drugs. NPs are broadly classified into various categories depending on their physical properties (e.g., morphology and size) and chemical characteristics. Nevertheless, they can be divided into four main categories: polymeric NPs (made of polymers like poly (lactic-coglycolic acid), dextran, and chitosan), metal NPs (e.g., gold, silver, and copper), ceramic NPs (e.g., calcium phosphates and mesoporous silica) and lipid-based NPs (mainly liposomes) (Fig. 2).

Fig. 2: Different types of nanoparticles used for osteosarcoma.

NPs used in osteosarcoma research are primarily categorized into four groups: polymeric NPs, metal NPs, ceramic NPs, and lipid-based NPs (mainly liposomes). Created with BioRender.com.

There are four primary routes for administering NPs: injection, inhalation, oral intake, and intravenous administration, with the latter being predominantly used in in vivo studies related to OS research^11,12,13,14, The Synthesis of NPs generally follows top-down or bottom-up protocols. Top-down methods involve breaking down larger materials into nanoparticles suitable for drug delivery, while bottom-up techniques entail the assembly of smaller building blocks to form larger particles¹⁵.

NPs categories

Polymeric NPs

Polymeric NPs range in size from 1 to 1000 nm and can be derived from either natural or synthetic polymers. Examples of natural polymers include chitosan, dextran, and gelatin, whereas synthetic polymers encompass materials like poly(ε-caprolactone) (PCL), poly (ethylene glycol) (PEG), and poly(lactide-co-glycolide) (PLGA)^16,17,

Natural polymeric NPs

The primary advantages of natural polymeric NPs lie in their availability in nature, biodegradability, biocompatibility, and low toxicity. However, they may pose immunogenicity concerns and often necessitate chemical modification prior to their utilization in NPs development¹⁵.

Keratin, a natural polymer, has unique tri-peptidic sequences such as “Arg–Gly–Asp” (RGD) and “Leu–Asp–Val” (LDV) sequences that bind to vitronectin integrin receptors, which are overexpressed by OS cells. Keratin was functionalized with the photosensitizer Chlorin-e6 and then loaded with the chemotherapeutic drug Paclitaxel (PTX) to create PTX-keratin NPs¹⁸. In vitro toxicity tests on OS cell lines in 2D and 3D systems showed that PTX and Ce6 have an additive effect. The combined cytostatic blockage of PTX and oxidative damage from reactive oxygen species (ROS) upon light irradiation had a superior effect compared to PTX or Ce6 alone. MTX is a widely used chemotherapy for the treatment of various cancers. Despite its efficacy in fighting cancer, MTX’s severe toxicity towards normal cells and low water solubility present limitations in its clinical use. To address these challenges, Li et al.¹⁹ developed poloxamer-modified trimethyl chitosan-encapsulated MTX. The resulting NPs showed increased cytotoxicity in human OS MG-63 cells in vitro when compared to free MTX due to improved cellular uptake.

Synthetic polymeric NPs

While natural polymeric NPs offer superior biocompatibility and lower toxicity compared to their synthetic polymeric NPs, synthetic polymeric NPs have garnered attention due to their stability and flexibility in obtaining polymeric materials. Moreover, the ability to synthesize these materials tailored to specific pathologies and patient needs underscores the advantages of synthetic polymeric NPs over natural ones²⁰.

NPs made of synthetic PLGA polymer are widely applied in drug delivery because of their biodegradable and biocompatible features. Irmak et al.²¹ encapsulated Salinomycin, an antibacterial and therapeutic agent known for its suppressive effects on the proliferation of different cancer stem cells, including OS, within PLGA NPs to address Salinomycin’s poor aqueous solubility. In vitro experiments utilizing an apoptosis assay revealed that PLGA NPs loaded with Salinomycin induced heightened apoptosis in OS MG-63 cells compared to unencapsulated Salinomycin, attributed to increased caspase-3 expression and decreased levels of c-myc and β-catenin.

In addition to drug delivery, PLGA and other polymers, such as PEG, are commonly utilized to modify nanocarriers to enhance stability, biocompatibility, and circulation time in the body^22,23,24, Notably, these polymers have been instrumental in addressing the challenge of initial burst release in NPs. For instance, Ray et al. ²⁵ developed LDH-MTX NPs by incorporating MTX with a Mg-Al- layered double hydroxide nanoceramic matrix. To prevent the initial burst release of MTX, they encapsulated the NPs in PLGA polymer. Results indicated that PLGA-LDH-MTX and PLGA-MTX NPs were both effective in suppressing tumor growth in subcutaneous mouse model compared to free MTX. PLGA-LDH-MTX was even more effective in terms of antitumor activity than bare MTX and PLGA-MTX, with a reduced likelihood of side effects when the same dose of MTX drug content was applied.

Metal NPs

In recent years, metal NPs, derived from bulk metals, have been extensively utilized in diagnostic imaging and drug delivery applications due to their physical and chemical properties, including mechanical strength, high surface area, thermal, optical, catalytic, and magnetic properties²⁶. Metal NPs can be classified into two main categories. The first category is NPs that are composed of pure metals, e.g., silver, copper, gold, titanium (Ti), platinum, zinc, magnesium (Mg), iron, and alginate NPs. The second category is metal oxide NPs such as iron oxide (magnetic NPs), Ti dioxide, silver oxide, zinc oxide, etc.²⁷.

Most metal oxide NPs have intrinsic anticancer properties, eliminating the need to load them with chemotherapy drugs^28,29,30,31, Pure metal NPs, especially gold NPs, are primarily used in photothermal therapy ^32,33, Additionally, metals have been reported as drug carriers in the treatment of OS. For instance, Popescu et al. ³⁴ utilized magnetite Fe3O4 NPs as carriers for Gemcitabine to enhance the cytotoxic effects of this chemotherapeutic agent on cancer cells, including OS MG-63 cell lines.

Superparamagnetic iron oxide NPs (SPIONs) are gaining widespread attention due to their biocompatibility and sensitivity to an applied magnetic field, yet unmodified simple SPIONs are prone to aggregation and unstable in biological conditions. Puiu et al. ³⁵ applied β-Cyclodextrin as a surface modifier for SPIONs to make them water-dispersible and to enable the loading of the hydrophobic anti-cancer drug PTX. The results from MTT assays showed that β-CD surface-modified SPIONs exhibited excellent biocompatibility, demonstrating no cytotoxic effects toward MC3T3-E1 murine osteoblasts. Meanwhile, the PTX-loaded β-CD surface-modified SPIONs significantly reduced the cell viability of the OS MG-63 cell line by 85%.

Ceramic NPs

Ceramic NPs are primarily comprised of carbides, oxides, carbonates, and phosphates of metals and metalloids. Examples of commonly used ceramic NPs include silica, alumina, titania, zirconia, calcium phosphates, calcium carbonate and hydroxyapatite (HAp). In recent years, remarkable progress has been made in the research on ceramic NPs for biomedical applications, especially in orthopedic and dental treatments, thanks to their unique processing, high mechanical strength, toughness and bioactivity^36,37,38,39.

Among various ceramic NPs, HAp NPs have been extensively studied in OS research due to their structural similarity to bone apatite, which enhances their biocompatibility and noninflammatory properties^{36,40,41,42,43,44} In their study, Zhou and his team⁴¹ developed Selenium-doped HAp nanowire/chitosan (Se-HAp/CSP) biopapers. When co-cultured with both cancerous and normal cells, the Se-HAp/CSP composite biopapers exhibited increased toxicity towards tumor cell lines compared to normal bone marrow stromal cells. Furthermore, anti-tumor experiments using a patient-derived xenograft tumor mouse model confirmed the ability of the Se-HAp/CSP biopapers to suppress tumor growth.

Calcium carbonate is another ceramic that is used in OS research^12,38,45,46, Li et al. ¹² developed organic-inorganic hybrid NPs based on calcium carbonate mineralization of polymers. These NPs offer several benefits, including pH sensitivity, small size, high biocompatibility, and biodegradability. Known as DOX-loaded calcium carbonate-crosslinked polypeptide NPs, they exhibited high drug loading capacity, improved cellular uptake, and increased cytotoxicity against mouse OS cells. Furthermore, they demonstrated enhanced antitumor efficacy and reduced side effects in subcutaneous and orthotopic OS mouse models.

The potential of ceramic NPs in sustained drug delivery has been hindered by their limited drug loading capacity, initial burst release, and short-term release. To overcome these challenges, researchers have explored the use of a combination of ceramic NPs with polymers as a strategy to enhance drug release and mechanical properties. Dan Son et al. ⁴⁷ investigated the use of calcium phosphate NPs containing anticancer drug in the presence of alginate as means to stabilize the nanocomposites. The resulting nanocomposites exhibited an initial burst release of the drug, followed by a slow and long-term release rate. Furthermore, the release profile was found to be dependent on the pH of the solution, with faster drug release observed at pH 4.5 compared to pH 7.4. Ghosh et al. ⁴² synthesized a novel nanocomposite by coating HAp NPs with DOX and further modifying them with chitosan. The purpose of this modification was to enhance the mechanical and biological properties of the nanocomposite. The results of their study showed that the HAp NPs alone were biocompatible with OS MG-63 cells, whereas the developed nanocomposite exhibited cytotoxicity towards OS MG-63 cells.

Liposomes NPs

Liposomes, the first FDA-approved therapeutic NPs, range in size from nanosized to micronized (20 nm up to several micrometers) and are closed vesicles with a bilayer structure. They are naturally produced by dispersing amphipathic molecules, such as phospholipids, in an aqueous environment. Liposomes have been extensively used as carriers of various drugs, including chemotherapeutics and antibiotics, thanks to their various advantages; these include the capability of holding both hydrophilic and lipophilic drugs, biocompatibility, low-immunogenicity, non-toxicity, and high drug encapsulation efficiency.

Additionally, the modification of liposomal surfaces is straightforward, and several strategies for their surface modification have been established, including polymer coating, altering surface charge, and modification with ligands^48,49,

The unique structure of liposomes, featuring a hydrophilic core surrounded by a phospholipid bilayer, allows the encapsulation of both lipophilic and hydrophilic drugs. Lipophilic drugs are integrated into the lipid bilayer, while hydrophilic drugs are entrapped in the inner space of the liposomal carrier, making liposomes ideally suited for the co-delivery of multiple drugs with different pharmacokinetic properties⁵⁰.

To enhance stability and prolong circulation half-time, liposomes can be coated with biocompatible hydrophilic polymers, most notably PEG, resulting in PEGylated liposomes. The PEG coating facilitates the escape of liposomes from the reticuloendothelial system (RES), thereby extending their circulation time in the bloodstream. Additionally, PEG reduces particle aggregation through steric hindrance stabilization, ultimately enhancing the stability of liposomes⁵¹. Haghiralsadat et al. ^52,53. demonstrated an approach to augment the cellular uptake of DOX in various OS cell lines by developing a PEGylated DOX-loaded liposomal formulation. This strategy led to increased intracellular uptake compared to free DOX. Consequently, assessments of cytotoxicity against OS MG-63 cell lines demonstrated that these nanoparticles displayed superior cytotoxic effects on MG-63 cells compared to free DOX.

Recent developments in systemic NPs-based drug delivery

NPs-based drug delivery systems have been shown to overcome several limitations associated with the existing therapeutic strategies and offer several advantages over systemic chemotherapy for the treatment of OS^12,18,54, These include efficiency in carrying high drug payloads, extending the drug’s half-life, minimizing toxicity, and effectively tackling issues related to drug solubility and stability. Recent advancements in nanoparticle-based drug delivery have focused on achieving more efficient and targeted drug delivery. This section will discuss these advancements, including targeted drug delivery, responsive drug delivery, and co-delivery systems.

Various NPs have been developed for targeted treatment of bone cancer to target the cancerous cells and minimize cytotoxicity to normal cells. In addition, nanocarriers with stimuli-responsive properties have been designed to release drugs in response to internal or external triggers, preventing premature drug release and ensuring precise drug delivery^45,55, Co-delivery drug systems enable the simultaneous administration of multiple agents, leading to synergistic improvements in treatment effectiveness and reduced drug resistance^38,56, (Fig. 3).

Fig. 3: Recent developments in systemic NPs-based drug delivery.

Recent advancements in NPs-based drug delivery include targeted drug delivery, stimulus-responsive drug delivery, and co-delivery of multiple drugs. Created with BioRender.com.

Targeted NPs

The commonly adopted approaches for targeting OS can be classified into three broad categories: organ-targeted therapy, cell-targeted therapy, and molecular-targeted therapy. In the realm of OS organ-targeted therapy, the most frequently employed strategy involves specifically binding to HAp, which is the principal component of bone tissue^{55,57,58,59,60}, (Table 1).

Table 1 Examples of osteosarcoma-targeted strategies used in drug delivery NPs

Bisphosphonates are commonly utilized as bone-targeting ligands due to their exceptional ability to bind to HAp. To enhance their therapeutic potential, bisphosphonate-functionalized HAp NPs loaded with the chemotherapeutic agent JQ1 have been developed⁵⁷. In vitro studies evaluating these NPs in both 2D, and 3D OS models have demonstrated their selectivity, as they showed increased toxicity towards murine OS K7M2 cells, compared to primary fibroblasts. Alendronate is another bone target ligand that belongs to the family of bisphosphonate. In a recent study⁵⁹, liposomes were modified with both alendronate and low molecular-weight heparin to facilitate the delivery of DOX. The resulting NPs were evaluated using orthotopic K7M2 OS and bone metastasis cancer models. The results showed remarkable suppression of tumor growth and inhibition of tumor metastasis. In another study⁶¹, nanoclusters decorated with alendronate and loaded with DOX were synthesized. The targeting ability of these nanoclusters was evaluated using an in vitro bone cancer model, and their affinity to HAp was assessed based on DOX binding efficiency. Results indicated that the targeted nanoclusters exhibited approximately five-fold higher affinity compared to untargeted nanoclusters. Furthermore, compared to unmodified nanoclusters, the developed nanoclusters demonstrated enhanced accumulation within tumors in an orthotopic mouse model with intratibial injection of human OS HOS/MNNG cells.

Aptamers are short, chemically synthesized, single‐stranded DNA and RNA oligonucleotides or polypeptide fragments that, upon folding, attain unique three-dimensional structures and bind to their target via structural recognition in a manner like an antibody-antigen interaction. Several aptamers have been developed against a variety of cancer targets, including extracellular ligands and cell surface proteins. These aptamers can serve as mediators between chemotherapeutics-loaded NPs and cancer cells. CD133 has been identified as a marker for cancer stem cells (CSC) in OS, resulting in the utilization of CD133 aptamers in OS-targeted drug delivery carriers^22,62,63, Epidermal growth factor receptor (EGFR) has been found to be overexpressed in OS cells. Accordingly, to achieve dual targeting for both OS CSC and cancer cells, Chen et al. ⁶² created NPs containing salinomycin labeled with both CD133 and EGFR aptamers. The results indicated that the targeted NPs showed increased cytotoxicity in both OS cells and CSC, surpassing the effectiveness of single targeting or non-targeted delivery methods. In vivo studies using a mouse model with subcutaneously injected human OS Saos-2 cells also showed that the administration of the targeted NPs resulted in the best inhibition of tumor growth in compared to other controls.

The molecular-targeted therapy is considered the most specific targeting strategy; it targets sites such as protein molecules, including receptors, peptides, or gene segments that are highly upregulated in tumor cells. Folic acid, Hyaluronic acid (HA), and peptides have been reported to be used as targeting ligands decorated in the nanocarriers for OS-targeted drug delivery systems. Recent studies have demonstrated the potential of utilizing the folate receptor for targeted drug delivery, as these receptors are frequently overexpressed in multiple cancers, including OS^64,65,66, Karimian et al. ⁶⁶ fabricated magnetic NPs loaded with DOX and grafted with folic acid, targeted against folate receptors. The results showed an enhanced targeted delivery and internalization of the modified NPs, leading to improved therapeutic effects of DOX on OS Saos-2 cells. HA, a biocompatible endogenous polysaccharide, acts as a key ligand for CD44 cell surface receptors, which are abundant in OS cells⁶⁷. To target these receptors, NPs, especially liposomes modified with HA, have been studied^67,68,69, A recent study by Gazzano et al. ⁶⁸ involved conjugating HA with H2S-releasing DOX-loaded liposomes. The results showed improved drug delivery and efficacy in vitro and compared to free drug and untargeted NPs. Peptides, including STP, RGD, and YSA, have been utilized to design NPs for targeted delivery to OS cells. STP has a specific affinity for vimentin, an overexpressed protein on the surface of various cancer cells, including OS cells^70,71, An STP-decorated disulfide crosslinked polypeptide nanogel was fabricated for the targeted delivery of shikonin⁷¹. These NPs selectively accumulated in orthotopic 143B OS tumors, specifically recognizing vimentin on the cell membrane. This resulted in significant antitumor efficacy and inhibition of pulmonary metastasis. The 12-amino acid peptide, YSA, functions as a ligand for the EphA2 receptor, a surface molecule that is highly upregulated in OS cells^72,73, In vitro studies⁷³ on DOX-loaded liposomes modified with YSA showed that the YSA-modified liposomes could efficiently target OS Saos-2 cells. This led to a higher therapeutic index of the prepared formulation compared to free DOX. RGD, a peptide with high cellular affinity, can bind to αvβ3 and αvβ5 integrins that are present on OS cells such as MG-63 and MNNG/HOS. In vitro studies⁷⁴ have shown that RGD-modified polymeric micelles have improved uptake into cells, specifically targeting OS MG-63 cells.

While targeted NPs offer great potential for treating OS, they face several challenges. For example, NPs designed to target OS using bisphosphonate ligands tend to accumulate in bone rather than specifically targeting malignant OS cells. Additionally, the extended presence of bisphosphonates in bone tissue may hinder osteoclast activity and disrupt bone homeostasis⁷⁵.

Furthermore, in the realm of cell- and molecular-targeted NPs for OS, most are still in the experimental phase. There’s a considerable transitional period before they can be clinically available due to numerous challenges. These challenges include the increased cost associated with ligand conjugation, potential alterations in NPs and ligand properties post-conjugation, and the need to identify suitable tumor markers or receptors for targeting.

Stimuli-responsive drug-releasing NPs

One of the prevalent challenges associated with conventional NPs is the premature release of their cargo before reaching their intended target site. Consequently, recent efforts have been dedicated to the design of modified nanocarriers capable of releasing their loaded drugs in response to various internal or external stimuli. Internal stimuli include differences in pH, redox potential, or enzyme concentrations. These triggers offer promising opportunities for responsive drug delivery systems, particularly in light of the distinct microenvironment found in tumor tissues compared to normal cells. Tumor microenvironments often feature acidic pH, elevated levels of glutathione (GSH) within the cellular cytoplasm, and the overexpression of specific enzymes. In addition to internal stimuli, external factors such as light, magnetic fields, temperature, and ultrasound have been explored. Various NPs systems, including liposomes, polymeric micelles, lipoplexes, and polyplexes, have been engineered to exploit these physical and chemical cues to fine-tune drug release. In the subsequent sections, we present an overview of the most extensively investigated environmentally responsive NPs^69,76,77,78, (Table 2).

Table 2 Examples of stimuli-responsive NPs used for osteosarcoma therapy

Redox-responsive NPs

GSH is a highly potent antioxidant and reducing agent in living cells. The concentration of GSH in the normal cellular cytoplasm is significantly higher than in extracellular fluid tissues (approximately 100-fold higher). Most remarkably, research has revealed that the concentration of GSH in cancer cells can be fourfold or more than that in normal cells⁶⁹. Thus, the introduction of redox-responsive agents, such as disulfide bonds, diselenide bonds, and ditelluride linkages, has enabled the development of redox-responsive NPs^79,80, These NPs can degrade within cancerous cells, facilitating the release of their cargo.

Disulfide bonds can be intracellularly cleaved by GSH in cancer cells, making them extensively used in the design of reduction-sensitive NPs. In research on redox-responsive NPs for OS, studies primarily focus on NPs with disulfide bonds^23,67,69,81, Yin et al. ²³ prepared reduction-responsive liposomes by attaching chitooligosaccharides (COS) to their surface through a disulfide linker. These reduction-sensitive liposomes, designated as Chol-SS-COS/DOX, exhibited stability under physiological conditions but underwent destabilization when exposed to reducing agents. Also, in the presence of GSH, the DOX release from Chol-SS-COS/DOX was accelerated compared to its release in the absence of GSH. In vitro, cytotoxicity study showed that reduction-sensitive liposomes showed higher cytotoxicity and more efficient internalization than non-reduction-sensitive liposomes in MG-63 cells. Moreover, the reduction-sensitive liposomes demonstrated a strong inhibitory effect on tumor growth in MG-63 cell-bearing nude mice and extended animal survival rates.

pH-responsive NPs

The pH in the bloodstream is around 7.4, while solid tumors including OS have an acidic extracellular environment with pH values ranging from 6.4–6.8^82,83, The acidic cellular environment exhibits even lower pH values, such as pH around 5.5–6.0 in endosomes and pH around 4.5–5.0 in lysosomes⁸⁴. The difference in the pH values between physiological conditions (e.g., blood) and tumor microenvironment, as well as between different cellular compartments, have been utilized for developing pH-responsive NPs that deliver drugs to the tumor tissues at both tissue and cellular levels.

Incorporating acid-sensitive linkers, such as acetal, hydrazone, glycerol ester, and amide groups, facilitates the conjugation of antitumor drugs to NPs. These linkers allow for the binding and stabilization of drugs to NPs under physiological pH conditions while subsequently permitting drug release within an acidic environment⁸⁵. Meshkini et al.⁸⁶ synthesized mesoporous zinc HAp (ZnHAp) decorated with a pluronic block copolymer, F127, and subsequently, they conjugated MTX onto the surface of the NPs through an amide bond. They investigated the release of MTX from the NPs under different pH values ranging from 4 to 7.4 in the presence of crude protease from bovine pancreas. The results revealed that a larger amount of MTX was released at pH 4 as it has the highest concentration of the active protease that’s most likely responsible for the cleavage of MTX from the NPs.

Calcium carbonate (CaCO₃) exhibits remarkable pH sensitivity as it undergoes dissociation in acidic solutions, making it a versatile choice for various controlled-release applications. CaCO₃ serves both as a nanocarrier for antitumor drugs and as a crosslinker between NPs and therapeutic agents^45,77, Fu et al. ⁷⁷ fabricated aragonite (CaCO₃ polymorph) NPs loaded with DOX. These NPs exhibited a pH-sensitive pattern, with 36% of DOX released in a PBS solution (pH 7.4) within the first 2 h and approximately 80% of DOX released within 24 h. While at acidic PBS buffer (pH 4.8), around 72% of DOX was released at the first hour, and about 90% was released within 8 h. Zhang et al. ⁴⁵ fabricated DOX-loaded HA NPs that were further crosslinked to CaCO₃, to enhance their stability and make the NPs sensitive to the acidic tumor microenvironment. They demonstrated that the amount of DOX released at pH 6.8 and 5.5 was 2.2 and 4.1 times higher than the neutral conditions (7.4), respectively. They also evaluated the biodistribution and antitumor efficacies of the developed NPs in both primary and advanced models of murine OS, compared with non-crosslinked NPs (HA-DOX) and free DOX, the HA-DOX/CaCO₃ showed effective tumor accumulation and highest tumor inhibition efficacy.

In acidic conditions, the amino groups present in chitosan can become protonated. This property enables chitosan to function as a pH-responsive polymer, which has been utilized in previous studies for the functionalization of NPs^76,87,88, Yang et al. ⁸⁸ prepared mesoporous ZSM-5 zeolites/chitosan core-shell nanodisks loaded with DOX (ZSM-5/CS/DOX). The CS layer on the nanodisks was used to regulate the DOX release, exhibiting pH-responsive behavior with a higher drug release rate in a slightly acidic environment compared to the neutral environment. Results demonstrated a pH-dependent release of DOX from the nanodisks. At normal physiological conditions (pH 7.4), the DOX cumulative release ratio from the ZSM-5/CS/DOX nanodisks was only 39.9% after 7 days of incubation. In contrast, when the pH decreased to 5.5, 71% of DOX was released. In addition, the therapeutic effects of the ZSM-5/CS/DOX nanodisks were studied with both and in vitro tests. Compared to the free DOX group, the mesoporous ZSM-5/CS/DOX group exhibited more effective MG-63 cells suppression with lower side effects.

The ionic interaction between the loaded drug and its carrier may provide a pH-sensitive property to the delivery system. Ahmadi et al. ⁸⁹ developed NPs consisting of a magnetic inner core and polymeric outer shell with cationic moieties to deliver MTX (MTX/Cat-MN) to the Saos-2 cells. The MTX release profiles from the nanocarriers were next evaluated under various pH solutions 5.0 and 7.4. At pH 7.4, the cumulative amount of released MTX from MTX/Cat-MN was 44.21 ± 2.98% after about 12.5 days. In contrast, under acidic conditions (pH 5.0), MTX/Cat-MN could release up to 72.73 ± 3.19% of total encapsulated MTX. Under low pH values, the carboxylate anion of MTX was protonated. This leads to the impairment of the electrostatic interactions between MTX and the Cat-MN protonated functional groups. Consequently, this can be the reason for the acceleration of the MTX release.

While certain pH-responsive or redox-responsive nanoparticles exhibit promising performance in vitro, they may be restricted by the complex physiological or pathological environment in vivo. Primarily, these responsive nanoparticles struggle to precisely regulate cargo release at tumor sites within optimal time frames and dosages. Moreover, their functionality may be impeded by the limited availability of stimulus factors in vivo. Additionally, the response kinetics in vivo are unpredictable due to the influence of various factors.

Light-responsive NPs

Light is an example of an external stimulus for drug delivery systems; it is quite beneficial due to its non-invasive nature and precise facilitation of spatiotemporal drug release. Light used in photo-responsive drug delivery nanocarriers can be categorized into three primary ranges: ultraviolet (100–400 nm), visible (400–700 nm), and near-infrared (700–1000 nm) light. Light within the 300–700 nm range is typically harnessed for applications involving superficial tissues, while near-infrared (NIR) light, with longer wavelengths, is employed for deeper tissue penetration⁹⁰.

Martínez-Carmona et al. ⁹¹ developed a visible light-responsive drug delivery system using mesoporous silica NPs loaded with topotecan, a chemotherapy drug. Porphyrin nanocaps blocked the pore outlets of the NPs via ROS-cleavable linkages. Upon exposure to visible light, the porphyrin nanocaps generated ROS that breaks the bonds and uncaps the pores, releasing 99% of the loaded drug after 18 h after being subjected to 30 min of low-intensity light irradiation. In vitro tests on OS HOS cells showed controlled release of topotecan and induced toxicity within the cancer cells. In another study, Chen et al. ⁹² created a DOX-conjugated polymer that can self-assemble into polymeric micelles (Poly-Dox-M) in water and is responsive to UV light. When exposed to UV light, the bond in the micellar polymer structure breaks, leading to a rapid release of DOX at the target tumor site. Recently, NIR-Responsive mesoporous silica-coated bismuth sulfide NPs encapsulating DOX were developed⁹³. These NPs demonstrated NIR-sensitive drug release, even under a very low power density of 0.3 W cm^–2.

The efficacy of light-responsive nanoparticles in treatment is impeded by the limited depth of light penetration. Visible and UV light (up to 700 nm) can only permeate biological tissues to a shallow extent, typically a few millimeters, due to strong scattering and absorption by soft tissues. Consequently, the utilization of Visible and UV light is confined to superficial tissues. While NIR light allows for deeper tissue penetration, reaching depths of up to a few centimeters, it may still fall short in reaching bone tissues situated deep within the body.

Co-delivery of drugs using NPs

Multidrug resistance (MDR) of tumors poses a major challenge to chemotherapy efficacy and contributes to cancer recurrence. This phenomenon is attributed to ATP-binding cassette (ABC) transporters such as P-glycoprotein, which efflux drugs from cancer cells. To overcome MDR, researchers have explored the use of polychemotherapy, which involves the concurrent administration of drugs with distinct mechanisms of action⁹⁴. Recently, the co-delivery of drugs via nanoplatforms has gained significant attention as a promising avenue for tackling MDR⁹⁵. Caliskan et al. ⁹⁶ developed a liposomal formulation co-encapsulating Gemcitabine (GEM) and Clofazimine (CLF). The hydrophilic core of the liposomes encapsulated GEM, an anticancer agent that disrupts DNA synthesis, while CLF, a lipophilic agent that inhibits Wnt signal transduction, was loaded in the lipid bilayer. This co-loading approach showed a synergistic effect in vitro, resulting in greater cytotoxicity than individual liposomal treatments of either GEM or CLF. A recent investigation⁹⁴ employed the formation of lipid-polymer hybrid NPs loaded with both DOX and Edelfosine. Evaluations demonstrated a marked enhancement in the anticancer efficacy of the dual drug-loaded NPs and suppression of tumor growth without any observable adverse effects.

Combination therapy is not restricted to chemotherapeutic agents alone. It can also involve a combination of chemotherapeutics and proteins or other therapeutic agents. Some proteins can offer unique advantages over chemotherapeutics, including inherent bioactivity, high functional specificity, and reduced side effects. This synergistic combination of chemotherapeutics and proteins can lead to improved therapeutic outcomes and reduced side effects in cancer treatment⁹⁷. A recent study³⁸ has demonstrated the development of calcium carbonate mineralized NPs loaded with the therapeutic protein cytochrome c and the chemotherapeutic agent DOX. The NPs were evaluated both in vitro using mouse OS K7 cells and in vivo with a K7 orthotopic OS mouse model. The results showed a significant improvement in biocompatibility and enhanced synergistic anti-OS effects compared to control groups. In a recent study, Hu et al. ⁹⁸ investigated the potential of selenium-doped calcium phosphate NPs loaded with DOX. Selenium, a trace element with potential antitumor effects, enhances the intracellular level of ROS and contributes to overcoming MDR by downregulating ABC transporter proteins, leading to the apoptosis of cancer cells. The developed NPs effectively downregulated the expression of MDR-related proteins in the ABC family and reversed MDR in OS cells. In vivo studies confirmed the efficacy of these NPs, demonstrating significant induction of tumor apoptosis, suppression of tumor growth, and reversal of MDR.

Zhang et al. ¹¹ created a crosslinked nanogel utilizing HA to co-deliver CDDP and DOX. Both drugs interact with DNA and cause damage, which is part of their anticancer mechanism. CDDP served not only as a supplementary anticarcinogen but also as a crosslinker, preventing premature release of DOX and thereby enhancing the synergistic therapeutic effects. The CDDP crosslinked DOX-loaded nanogel exhibited optimized biodistribution, elevated antitumor efficacy, and reduced multi-organ toxicity compared to free drugs and their combination using a K7 subcutaneous OS mouse model.

Limitations and challenges

As discussed previously, NPs-based drug delivery systems have emerged as a promising approach to address the limitations of systemic drug administration and have made significant progress in paving their way into clinical practice. These NPs offer distinct advantages over traditional chemotherapy, including a significant reduction in adverse effects, improvements in drug pharmacokinetics, and extended circulation time in the bloodstream^99,100, Despite the substantial progress made in developing NPs for cancer treatment, several challenges and limitations persist. NPs are typically administered intravenously to ensure direct access to the bloodstream; however, this method often results in rapid NPs clearance, limiting their effective interaction with target sites. More than 95% of systemically injected drugs tend to accumulate in organs such as the liver, spleen, and lungs, with less than 5% reaching the targeted tumor site, even with advanced strategies like coupling targeting ligands or designing stimuli-responsive release mechanisms^101,102, This challenge is even more pronounced in delivering drugs to bone tissue due to the highly mineralized extracellular matrix of bone, which impedes the diffusion of molecules from the bloodstream into the bone tissue. Consequently, higher NP dosages are required, leading to increased drug concentrations and potentially greater toxicity¹⁰³.

Local drug delivery

Recently, local administration of chemotherapy approaches has drawn much attention, offering significant benefits over systemic drug delivery, especially for bone cancer treatment. This strategy bypasses the limitations that impede the NPs in the bloodstream before reaching the target site. These limitations include short blood circulation time, clearance by Kupffer cells, low local concentrations, and limited accumulation in target tissue. By directly delivering drugs locally to the target site, local administration enables higher drug concentrations at the desired location, resulting in lower systemic toxicity and enhanced therapeutic efficiency.

Local delivery of chemotherapeutics can be achieved through different approaches, such as implanting nanocarriers, injecting hydrogels, and implanting scaffolds (see Fig. 4).

Fig. 4: Common strategies for local delivery of anticancer agents.

Various approaches for local delivery of chemotherapeutics include the use of implantable nanocarriers, injectable hydrogels, and implantable scaffolds. Created with BioRender.com.

Implantable nanocarriers

To enhance the efficiency of NPs delivery, recent research has shifted from intravenous injection to local delivery directly at the tumor site. This enhancement is achieved by implanting the NPs in a precise and localized intratumoral delivery manner^{44,104,105,106}, In OS studies, implantable NPs primarily involve HAp NPs injected locally near the tumor in murine models.

A ternary nanodrug delivery system consisting of HAp, bovine serum albumin (BSA), and the chemotherapy agent PTX was prepared for the locoregional treatment of OS¹⁰⁴. The efficacy of this complex was evaluated using an orthotopic OS mouse model. Results showed that the ternary NPs exhibited superior anticancer effects compared to control groups, which consisted of bare PTX drug and HAp-BSA NPs. Additionally, the ternary nanodrug delivery system effectively inhibited the metastasis of the tumor to other organs. Recently, Liu and his team⁴⁴ demonstrated the efficacy of local delivery of DOX via implantable HAp particles. In a mouse model of aggressive OS induced by subcutaneous injection of aggressive human OS 143B cells, they demonstrated that the local delivery of DOX via HAp particles resulted in a more pronounced tumor eradication effect compared to systemic administration (Fig. 5). Additionally, combining micro HAp particles in a carrier with nano HAp particles improved biocompatibility and safety by ensuring that the nano Hap particles were retained locally within the tumor site.

Fig. 5: Comparing the efficiency of DOX delivery through local HAp particles and systemic injection.

A schematic illustration showing the efficiency of doxorubicin (DOX) delivery via locally implanted HAp particles compared to systemic injection. Intravenous injection results in only 5–10% of DOX accumulating in the tumor, leading to limited therapeutic effect. In contrast, local delivery through HAp particles achieves a stronger tumor eradication effect. (Reprinted with permission from ref. ⁴⁴).

Injectable hydrogels

The utilization of hydrogel-mediated local drug delivery systems has gained increasing interest in recent years due to several advantages. These systems exhibit ease of injectability and a mild gelation process, as well as reduced systemic toxicity. Moreover, hydrogels can be administered directly at the tumor site via a syringe, simplifying the delivery process. Their highly porous structure can be finely tuned by adjusting the density of cross-links in the gel matrix and the hydrogels’ affinity for the aqueous environment in which they are swollen. This porosity facilitates the incorporation of drugs into the gel matrix through straightforward physical mixing. The release of drugs from hydrogels depends on factors such as water content, pore size, and the diffusion coefficient of the drug molecules. Typically, hydrogels with high water content and large pore sizes exhibit relatively rapid drug release, spanning from a few hours to several days¹⁰⁷.

Triblock copolymer PLGA-PEG-PLGA, an injectable thermosensitive hydrogel, has gained widespread recognition as a potent drug local delivery vehicle due to its good injectability, biodegradability, and biocompatibility^108,109, In a study by Yang et al. ¹⁰⁹, the efficacy of a PLGA-PEG-PLGA-based hydrogel as a delivery vehicle for DOX was investigated in a K7 subcutaneous OS mouse model. The results indicated a remarkable improvement in the maximum tolerated dose of DOX, which was two-fold higher than that of the intravenous injection of free DOX. Additionally, the localized administration of DOX via the hydrogel did not result in obvious systemic toxicity. In a recent study by the same group¹¹⁰, the efficacy of a PLGA-PEG-PLGA hydrogel was evaluated as a carrier for the delivery of a β cyclodextrin curcumin (CD-CUR) inclusion complex and DOX. The results obtained from the K7 subcutaneous OS mouse model indicated that the co-loaded hydrogel had a superior antitumor effect compared to free DOX + CD-CUR or single-drug treatments. Moreover, the localized treatment strategies demonstrated high systemic safety. In another study, Si et al. ¹¹¹ synthesized PLGA-PEG-PLGA hydrogel encapsulated with both DOX and CDDP. The drug release profile from the hydrogel exhibited an initial burst release during the first two days, followed by a sustained release for more than 10 days. In a mice OS Saos-2 xenografts model, the co-loaded hydrogel exhibited superior efficiency in suppressing tumor growth compared to the administration of either the free drugs or single drug-loaded hydrogel, suggesting a synergistic anti-tumor effect. Importantly, the localized administration of different treatments did not result in any detrimental impact on other vital organs, indicating a low systemic toxicity.

Zheng and colleagues¹¹² introduced a hydrogel-based delivery system for the local administration of Combretastatin A-4, an antiangiogenic agent, and docetaxel, a chemotherapy agent. The system consists of injectable thermosensitive polypeptide hydrogel embedded with PLGA microspheres. Results from the K7 subcutaneous OS mouse model demonstrated that this co-loaded hydrogel effectively suppressed tumor growth, surpassing the efficacy of other formulations, including free drugs, a combination of free drugs, and single-drug-loaded hydrogels. Furthermore, through histopathology and immunohistochemistry analysis, the co-loaded hydrogel demonstrated a significant enhancement of antiproliferative effects and reduction of drug toxicity.

Hydrogels, despite their promising results in treating OS, face challenges due to their low tensile strength. This limitation hinders their use in load-bearing applications and can lead to premature dissolution or displacement from a targeted local site. Furthermore, hydrogels often fail because of poor mechanical and structural stability, which prevents them from maintaining complex structures.

Scaffolds

The surgical resection of a bone tumor often leads to the formation of critical-sized bone defects that do not possess the innate capacity for spontaneous healing. Consequently, following the removal of a bone tumor, clinicians are thus faced with the challenge of addressing these defects, typically resorting the defect by implantation of biomaterial scaffolds. However, the conventional scaffolds employed in this context lack therapeutic capabilities to eradicate residual tumor cells. In light of this, there has been a growing interest in the development of localized drug delivery systems integrated within bioactive scaffolds¹¹³. These scaffolds not only aim to fill the post-surgical bone defect but also ensure the targeted local delivery of a high local concentration of chemotherapy. They are predominantly constructed from materials such as metal, bioceramics, biodegradable polymers, and composites.

Drug loaded -scaffolds

Metal

Metals, including Ti and its alloys, stainless steels, Mg alloys, and other biodegradable metals, are primarily recognized for their exceptional mechanical strength. Titanium is extensively utilized in clinical applications, particularly for dental implants, and is also a preferred material for load-bearing applications^113,114,115, However, Titanium implants often face challenges with poor osseointegration (lack of tissue adherence), leading to implant failure. One approach to address this issue is by manipulating the implant’s surface topography. For example, Maher et al. ¹¹⁴ fabricated titanium implants with dual micro- and nanotopography to enhance bone integration. Additionally, these implants were loaded with two types of drugs, DOX and apoptosis-inducing ligands (Apo2L/TRAIL). In vitro studies demonstrated the implants’ strong anticancer efficacy against cancer cells.

Bioceramics

Currently, bioceramic scaffolds, including materials such as calcium phosphates (specifically HAp and TCP), bioactive glasses, and silicate-based bioceramics, are widely used in bone-related clinical settings. Their popularity stems from their ability to closely mimic the mineral phase of bone, making them conducive to excellent bone regeneration^116,117, However, the main drawback of bioceramics is their intrinsic brittleness, which means they cannot withstand deformation without rupturing. This brittleness can lead to the sudden failure of the scaffold structure under load-bearing conditions.

Several recent studies have explored the use of polymers, such as PLGA, PCL, chitosan, and cyclodextrins, in conjunction with bioceramic bone scaffolds. These polymers are employed to regulate the release kinetics and mitigate the burst release of biomolecules or chemotherapeutics from the scaffolds^118,119, In one notable investigation, Bischoff et al. ¹¹⁸ employed a coating of cyclodextrins on HAp, which served as a carrier for DOX. The findings revealed that the DOX released from the cyclodextrin-coated HAp demonstrated sustained biological activity, leading to prolonged and enhanced cytotoxic effects on both OS MG-63 cells and non-malignant cells (primary osteoblasts and endothelial cells) compared to biomaterials lacking cyclodextrin-loaded DOX. Furthermore, they evaluated the delivery system in a hypoxic environment (less than 1% O₂), mimicking the initial conditions post-bone resection. The drug delivery system effectively inhibited cancer cell growth while preserving the viability and proliferation of healthy cells. In another study¹²⁰, researchers employed 3D printing to create calcium phosphate cement (CPC) scaffolds with interconnected pores. These scaffolds were further enhanced by applying a stable coating of a model anticancer drug, 5-fluorouracil, using a combination of hydrophilic solutions of Soluplus and PEG polymer. Dissolution studies conducted in vitro demonstrated that nearly 100% of the drug was released within a span of 2 h, indicating a rapid release rate. Additionally, cell culture experiments utilizing two distinct cell lines exhibited a significant inhibition of cell growth, as evidenced by a significant reduction in cell numbers after a period of 5 days.

Polymers

Polymeric scaffolds can be made from either natural or synthetic polymers. Natural polymers include silk fibroin, collagen, gelatin, and fibrin. These polymers offer advantages such as biodegradability and bioactive properties, which enhance cellular interaction and performance within biological systems¹²¹. However, natural polymers are suboptimal for bone applications due to their low mechanical stability and lack of tunability in degradation rates.

Among synthetic polymers, PCL, polylactic acid (PLA), PLGA, and their derivatives are the most used. These materials are favored for their controllable biodegradability and ability to generate porous scaffolds. Biodegradable synthetic polymers can be synthesized through the manipulation and customization of their molecular structure. This biodegradability is achieved through molecular design, where certain polymers incorporate chemical bonds susceptible to hydrolysis when exposed to the body’s aqueous environment¹²². Alternatively, some polymers degrade through cellular or enzymatic pathways. However, synthetic polymers also have limitations, such as the production of high local concentrations of acidic degradation products and the loss of mechanical strength after degradation.

In a recent study, Wang and colleagues¹²³ developed 3D biodegradable printed PLLA implants that were loaded with DOX, ifosfamide, and MTX for the treatment of OS. The drug delivery system exhibited several advantages, including localized chemotherapy, the capacity to deliver multiple drugs, and sustained drug release over an extended period.

Scaffolds containing drug-loaded NPs

Recent studies have explored the incorporation of drug-loaded NPs into scaffolds. This approach offers distinct advantages, primarily enhancing drug potency and stability while facilitating a controlled release of the encapsulated drug. Loading drugs onto NPs not only optimizes their efficacy but also ensures a more sustained and targeted delivery, contributing to the overall efficacy of the therapeutic intervention.

Liposomes have been commonly utilized as nanocarriers in numerous studies^124,125, In a particular study¹²⁵, liposomes loaded with Ruthenium were integrated into a PCL scaffold. The findings demonstrated a relatively prolonged drug release over a 48-hour period. Furthermore, MTT assays were employed to assess MG-63 cell viability and apoptosis rates. The experimental results indicate that Ruthenium induces mitochondrial dysfunction, leading to apoptosis in MG-63 cells.

In a recent study¹²⁶, the researchers explored the utilization of PLGA NPs loaded with DOX, which were then incorporated into CPC. The objective was to enhance the localized delivery of DOX and evaluate its cytotoxic effects on U2OS cells, both in monoculture and coculture with mesenchymal stem cells, in comparison to CPCs solely loaded with DOX. The findings revealed that the inclusion of PLGA-DOX NPs within the CPC matrix resulted in a more efficient release of DOX (80% of DOX released over 7 days), leading to elevated cytotoxicity levels in U2OS cells.

Jiang et al. ¹²⁷ have developed 3D-printed gelatin-based scaffolds using a combination of polydopamine (PDA)-hybridized nanosized zeolitic imidazolate framework-8 (pZIF-8 nanoMOFs) and PDA-decorated-hydroxyapatite NPs. In both in vitro and studies, the researchers examined the effects of encapsulating the nanoMOFs with anti-cancer CDDP into the scaffolds. The findings demonstrated that the encapsulation of CDDP effectively inhibited tumor growth in a subcutaneous OS mouse model. Release studies have demonstrated that CDDP is released in response to the tumor microenvironment due to the pH and H₂O₂ sensitivity of pZIF-8 nanoMOFs (with 50% of CDDP released after 5 days).

Multifunctional scaffolds

Multifunctional scaffold-based drug delivery systems

Common bone scaffolds have served a dual purpose by efficiently filling critical-sized bone defects resulting from surgical bone tumor resection and also functioning as a reliable delivery system for chemotherapy and therapeutic agents. In contrast, the newly introduced multifunctional bone substitutes not only adeptly address filling and delivering but also introduce an additional function: actively promoting bone regeneration. This advanced category serves a triple purpose, addressing the following key aspects:

• 1. Fill- filling the critical-sized bone defect resulting from surgical restriction of the bone tumor.

• 2. Deliver – local administration of chemotherapeutic drugs, facilitating the elimination of any remaining tumor cells after surgery while minimizing potential side effects.

• 3. Repair – promoting bone regeneration and restoring bone defects.

For a scaffold to effectively promote bone regeneration, it should mimic the structure and function of natural bone and possess the following essential properties:

• 1. Biocompatibility: the scaffold must integrate with the native bone without inducing inflammatory reactions.

• 2. Mechanical strength: it should have mechanical properties that match those of the host tissue, providing adequate support^128,129,130,

• 3. Biodegradability: the degradation rate of the scaffold should be synchronized with the bone regeneration process¹³¹.

• 4. High porosity and interconnectivity: a porosity of over 60%, with interconnected pores ranging from 100–500 μm, is optimal for encouraging cell attachment, migration, and ingrowth throughout the scaffold¹³².

• 5. Surface functional characteristics: the scaffold should have surface properties that promote cell adhesion and proliferation.

• 6. Osteoconductivity and osteoinductivity: osteoconductive scaffolds support the migration of bone-related cells, such as mesenchymal cells, osteoblasts, and osteoclasts. Osteoinductive scaffolds induce the differentiation of various cell types into osteogenic cells¹³³.

Table 3 provides a summary of the latest scaffold developments used as delivery systems for OS treatment, highlighting their demonstrated potential in facilitating bone regeneration.

Table 3 Scaffolds based on drug delivery system for osteosarcoma treatment and their role in bone regeneration

Several in vitro studies have introduced multifunctional scaffolds^{124,134,135,136,137}, In one such study¹³⁵, researchers engineered a chitosan/nHAp scaffold loaded with zoledronic acid (CS/nHAp/Zol). The resultant CS/nHAp/Zol scaffolds demonstrated the inhibition of giant cell tumors of bone growth in vitro. In addition, these scaffolds exhibited minimal toxicity toward human bone marrow mesenchymal stem cells. Furthermore, the CS/nHA/Zol scaffolds displayed osteoinductivity comparable to that of the CS/nHA scaffolds. In a separate study, Tan et al. ¹³⁴ developed a composite scaffold comprising PLLA and nHAp, encapsulating the drug metformin. Surprisingly, metformin demonstrated dual functionality, acting as a tumor cell suppressor and a bone regeneration accelerator, depending on its concentration. The scaffolds effectively inhibited the proliferation of Saos-2 cells through apoptosis induction. Additionally, these scaffolds supported human bone marrow mesenchymal stem cell adhesion and proliferation, fostering osteogenesis throughout prolonged culture periods. In another study, Bose and colleagues¹¹⁹ coated a TCP scaffold with PCL to evaluate the controlled release of vitamin C from the scaffold. The results revealed that the presence of PCL helps to reduce the burst release of vitamin C from TCP scaffolds during the first 24 h of release, leading to a more controlled and sustained release of vitamin C over 60 days. These scaffolds were found to enhance the proliferation, viability, and differentiation of human fetal osteoblast cells. Furthermore, preliminary in vitro study demonstrated that these scaffolds also inhibited the proliferation of OS cells.

In an in vivo study, Lu et al. ¹³⁸ developed PDA- coated composite scaffold consisting of DOX-loaded lamellar HAp and PLGA (PDA@DH/PLGA). The PDA@DH/PLGA scaffold not only exhibited a significant inhibitory effect on OS MG-63 cell growth but also demonstrated enhanced adhesion and proliferation of osteoblasts during a 20-day drug release period. Preliminary assessment of in vivo osteogenesis using a mouse skull defect model revealed superior bone growth surrounding the PDA@DH/PLGA scaffold compared to the DH/PLGA scaffold without PDA coating after 20 days of drug release. In another in vivo study, Zhang et al. ¹¹⁵ engineered a porous titanium scaffold loaded with nano-HAp and implanted it in a critical-sized segmental bone defect within a VX2 tumor environment in a rabbit tumor model. The nano-HAp-releasing scaffold exhibited high efficacy in suppressing tumor growth and osteolytic lesions, concurrently promoting bone regeneration (Fig. 6).

Fig. 6: Antitumor and segmental bone defect healing ability of n-HA–loaded titanium scaffold.

a Diagram depicting the preparation of tumor cell suspension and seeding into scaffold. b Implantation of tumor cell–seeded scaffold at segmental bone defect of rabbit femur. c Tumor volume of the rabbits implanted with empty scaffolds or n-HA/scaffolds within 5 weeks. Error bars represent SD. n = 4 per group. **P < 0.01 compared to empty scaffold, two-way t test. d Photographs of excised implants with tumor from weeks 2 to 5. e Micro-CT–reconstructed images of the implants and adjacent bone tissue. B, bone; S, scaffold; arrows show adjacent cortical bone resorption by tumor. f Histological observation of the implanted scaffolds. S, scaffold; T, tumor; red arrows indicate new bone formation. (Adapted with permission from ref. ¹¹⁵).

Multifunctional scaffold based on synergistic therapy

In addition to drug-loaded scaffolds, another approach for addressing tumor cell residues and repairing bone defects involves photothermal therapy or magnetic fluid hyperthermia functionalized scaffolds^{139,140,141,142}, Unlike drug-loaded scaffolds, these functionalized scaffolds utilize the high temperature generated by photothermal agents or magnetic NPs to eradicate tumor cells.

In photothermal therapy, the scaffolds contain photothermal agents that absorb NIR light radiation, converting it into localized heat. This elevated temperature, reaching up to 50 °C, effectively destroys tumorous cells. Conversely, in a magnetic fluid hyperthermia-based approach, the scaffold incorporates magnetic materials or introduces magnetic agents to a non-magnetic scaffold. The application of an external magnetic field generates heat locally within the bone defect. Studies have shown that temperatures ranging from 42 to 46 °C (moderate hyperthermia) are promising in bone cancer therapy, effectively eliminating cancerous cells without harming surrounding healthy tissue^143,144,145,

Recent research has introduced a synergistic therapy combining drug-loaded scaffolds with photothermal therapy or magnetic fluid hyperthermia. This approach aims to achieve controlled release of the loaded anti-tumor drug upon increased temperature, providing an on-demand treatment strategy.

Yang et al.¹⁴⁶ developed porous scaffolds comprised of magnetic mesoporous calcium silicate/chitosan (MCSC) for synergistic chemo-photothermal therapy (Fig. 7). Their findings demonstrate that NIR laser stimulation elevates temperature and facilitates DOX release from MCSC scaffolds, with a 24-hour cumulative release ratio of 79.3%, suggesting rapid localized delivery of anti-tumor agents to combat malignancy. In a subcutaneous OS mouse model, MCSC/DOX scaffolds significantly suppressed tumor proliferation compared to MCSC alone. Additionally, MCSC scaffolds without DOX were evaluated in a critical-sized calvarial-defect rat model, revealing their capability to promote in vivo bone formation. In another study, Wang et al.¹⁴⁷ developed cryogenic 3D printed porous scaffolds incorporating 2D black phosphorus nanosheets, DOX, and osteogenic peptide, serving as the photothermal agent, anti-cancer medication, and osteogenic factor, respectively. Employing these scaffolds achieved tumor cell elimination and long-term prevention of tumor recurrence in a subcutaneous OS mouse model through on-demand photothermal therapy and sustained localized DOX release at a low concentration. In vivo outcomes indicated that photothermal therapy alone inadequately eliminates residual tumor cells, whereas sustained DOX release at low concentrations suppresses recurrence. In a separate cranial defect rat model, the scaffolds, initially irradiated by NIR laser for 10 min to mimic the anti-cancer stage, exhibited enhanced in vivo bone regeneration in cranial defects, attributable to the synergistic effects of the bony environment and sustained peptide release.

Fig. 7: Schematic illustration of MCSC scaffolds and their multi-functions.

The MCSC scaffolds exhibit excellent properties in drug delivery and simultaneously improve the efficacy of photothermal therapy under the irradiation of NIR laser. Additionally, the MCSC scaffolds also enhance new bone regeneration effectively by promoting osteogenic differentiation. NIR, near-infrared. (Reprinted with permission from ref. ¹⁴⁷).

A preliminary study by Farzin et al.¹⁴⁸ explores combining magnetic fluid hyperthermia therapy with local drug delivery using a scaffold made of hardystonite loaded with Fe ions and CDDP. Further research is needed to understand how magnetically induced heating and drug delivery via these scaffolds affect cancer cell behavior and in vivo osteogenesis.

Outlook

Osteosarcoma, the primary bone malignancy affecting children and young adults, poses significant challenges due to its malignancy, invasiveness, rapid progression, metastatic potential, and high mortality rate. The standard therapy, which involves surgery and chemotherapy, achieves long-term survival in over 60% of localized cases¹⁴⁹. However, this approach is hindered by drawbacks such as toxicity, side effects, recurrence, drug resistance, and rapid blood clearance.

This review paper explores the potential of systemic NPs delivery for chemotherapy/therapeutic agents to enhance efficacy and minimize side effects. Despite exciting developments, challenges such as high clearance and accumulation in the liver, low tumor site accumulation, especially in bone tumors, and concerns about long-term biosafety, particularly for metal and ceramic NPs, remain inadequately assessed^150,151,

Subsequently, we investigated the use of local-based drug delivery, with a specific focus on scaffold-based drug delivery. This approach serves a dual purpose: it effectively targets residual cancerous cells in the bone post-surgery through the localized administration of chemotherapeutic drugs, and it facilitates the eradication of any remaining tumor cells after surgery while minimizing potential side effects. Additionally, this system addresses the challenge of filling critical-sized bone defects resulting from the surgical removal of the bone tumor.

We then explored a new class of scaffolds, namely multifunctional scaffolds, which serve a third function beyond the dual purposes mentioned: they have been shown to induce bone regeneration. Despite advancements in recent studies on multifunctional scaffolds, limitations still exist. Firstly, studies that have developed scaffolds encapsulated with chemotherapeutic drugs have not examined the impact of the loaded therapeutic agent on healthy cells and osteogenesis^{136,138,146,147}, The bone regeneration capacity of the scaffold has been tested without encapsulating the drugs or after releasing the chemotherapeutic drug, and then osteogenesis is assessed using in vitro or in vivo. This approach does not accurately represent the real bone tumor environment, where healthy cells are also affected by the high local concentration of chemotherapeutics. The toxicity of these drugs may hinder bone regeneration; thus, it is important to examine the effect of therapeutic agents on bone regeneration.

Secondly, the models used to examine the anticancer effectiveness of the scaffold often rely on ectopic tumor models, primarily at subcutaneous sites, which lack physiological and anatomical similarity to clinical conditions, thereby providing limited predictive value for clinical outcomes. Orthotopic models, which involve the implantation of tumor cell lines or patient-derived cell xenografts into the bone, provide a more reliable replication of the original tumor site. However, they present notable hurdles and technical complexities, particularly in mouse models, due to bone size constraints and challenges in scaffold insertion and tumor growth control¹⁵². A recent study has demonstrated the successful development of an orthotopic humanized tissue-engineered OS model in a genetically modified X-linked severe combined immunodeficient (X-SCID) rat¹⁵³. Using an orthopedic OS model in rats offers many advantages over mice, mainly because their larger size allows the development of larger tumors, which supports the conduction of clinically relevant diagnostic procedures such as medical imaging and properly executed biopsies. Additionally, the larger femur size in rats allows proper resection with appropriate margins, including a healthy tissue zone to ensure full tumor resection and subsequent filling with biomaterials.

Thirdly, identifying relevant preclinical animal models for the simultaneous examination of anti-tumor efficacy and bone regeneration remains challenging. Typically, studies address these aspects separately, with immunosuppressed rodent models preferred for bone malignancy and larger models, such as rabbits and rats, favored for bone regeneration^154,155, (Fig. 8). So far, only one study has reported testing the antitumor and bone regeneration ability of the scaffold simultaneously¹¹⁵. This was achieved by establishing a rabbit femur bone tumor model with VX2 tumor cells obtained from a tumor-bearing rabbit. While this model successfully demonstrated segmental bone defect repair after tumor resection, the use of human cell lines rather than rabbit cells remains crucial for ensuring translational relevance. Human cell lines offer a closer approximation to human disease, improving the reliability and applicability of research findings to human clinical settings.

Fig. 8: Commonly employed animal models in the study of multifunctional scaffolds for anticancer therapy and bone tissue regeneration.

commonly used animal models in studies of multifunctional scaffolds are depicted. Typically, these studies examine anti-tumor efficacy and bone regeneration separately, with immunosuppressed rodent models preferred for bone malignancy and larger models, such as rabbits and rats, favored for bone regeneration. Created with BioRender.com.

To simultaneously evaluate scaffold functionality for bone defect repair and antitumor efficacy, two approaches are proposed:

• 1. Creating a bone defect and then implanting the scaffold with tumor cells into the defect site.

• 2. Transplanting OS cells into the bone via pretibial/intraosseous injection, followed by excising the developed tumor and implanting the scaffold.

A summary of potential animal models capable of assessing both anticancer efficacy and bone regeneration is presented in Table 4. Establishing more relevant models is crucial due to the variation in mineral density, mechanical cues, and biochemical composition of bones across different species, including humans, sheep, mice, and rats. Therefore, success in animal models may not necessarily be directly translated to human patients. Consequently, bone substitutes that demonstrate efficacy in animal models may not necessarily be directly applicable to human patients, widening the gap between animal models and clinical application.

Table 4 Potential animal models for simultaneous assessment of tumor suppression and bone regeneration