A set of diseases collectively referred to as “cancer” are those in which uncontrolled tissue growth manifests malignantly, leaving cells without their usual form and/or functionality ^[1]. These abnormal cells can infiltrate other bodily areas, multiply, undergo angiogenesis, trick the immune system, and cause life-threatening malignancies ^[2]. Timely and proper therapy is mandated to prevent the unchecked growth of tumors and their impact on cancer patients. Cancer treatment is challenging, nevertheless, partly because of the unchecked metastatic pathways in invasion, neovascularization, circulation, extravasation, and migration, as well as the tumor cells’ increasing resistance to chemotherapeutic drugs ^[3].

Radiation therapy, surgery, hyperthermia, and stem cell therapy are examples of non-pharmacologic treatments that can be used. Combinatorial techniques between two or more therapeutic choices can also be sought after [4,5]^[4][5]. Intrusiveness, low drug solubility, brief circulation time of chemotherapeutics, multidrug resistance, indiscriminate targeting, and off-target adverse effects are a few of each approach’s drawbacks ^[6]. As a result, despite the variety of approaches that are already accessible, there is still a need to design safer and more effective therapeutic approaches. Additionally, advances in diagnosis and imaging methods would enable quicker cancer detection and better tracking of the disease’s trajectory, enabling us to comprehend the condition’s progression and tailor the appropriate treatment modalities accordingly.

Nanomedicine has emerged as a practical interdisciplinary approach for better managing human well-being, including the search for efficient anticancer therapies, to bridge this gap [7,8]^[7][8]. Nanotechnology offers a wide range of advantages by offering the conventional paradigms better localized therapeutic efficacy, less systemic toxicity, improved diagnostic sensitivity, and enhanced imaging capabilities [9,10,11,12,13]^{[9][10][11][12][13]}. It is recognized as a workable drug delivery channel for chemotherapeutic entrapped nanoparticle-based therapy due to its capacity to circumvent multidrug resistance and encapsulate and shuttle molecules with different physicochemical attributes to the desired tumor site ^[14]. Experts worldwide focus on the field mentioned above owing to the multiple benefits nanoformulations give over conventional therapeutics. For the targeted distribution of drugs for a multitude of therapeutic applications, a variety of nanocarriers have been used, including polymeric nanoparticles, nanoemulsions, liposomes, nanostructured lipid carriers, solid lipid nanoparticles, metallic nanoparticles, nano drug conjugates, dendrimers, hydrogels, carbon nanotube, and many others (Figure 1) [15,16,17,18]^{[15][16][17][18]}.

Developing bioactive compounds that can be conjugated or encapsulated and released when exposed to the tumor microenvironment’s specific chemical and biological circumstances has received particular attention in research investigations. These approaches are termed passive and active targeting strategies, enabling the suitable delivery of chemotherapeutics at the targeted tumor site (Figure 2). These nanocarriers can increase a drug’s bioavailability, aggregate in a tumor effectively, stimulate tumor cell internalization, couple therapeutic compounds with imaging tools, and enhance antitumor activity ^[19].

Various research demonstrates topotecan’s anticancer properties, employing various nanoparticle-based delivery systems among the several camptothecin derivatives ^[21]. Topotecan is a topoisomerase 1 (TOP1) inhibitor. The chemical structure of topotecan is depicted in Figure 3. The vital enzyme TOP1 is found in higher eukaryotes and is crucial for DNA replication. DNA, a supercoiled double helix, untangles during the replication process to produce single strands that serve as a framework for synthesizing novel strands. When the helix starts to unravel, TOP1 joins DNA in a momentary cleavable complex and creates nicks in the DNA to reduce torsional stress. The enzyme is released owing to a transitory complex, enabling the new strand to be re-ligated ^[22].

2. Passive Targeted Delivery Approach for Topotecan

The purpose of passive targeting is to take advantage of the differences between tumor and normal tissues. Chemotherapeutics are efficiently transported to the target site by passive targeting to accomplish a therapeutic function. Substantial cancer cell multiplication causes neovascularization and wide fenestrations in the vascular wall, enhancing the tumor vessels’ permeability relative to healthy vessels [33]^[23]. Macromolecules, such as NPs, might escape from blood arteries supplying the tumor and amass within tumor tissue due to the fast and deficient angiogenesis. The accumulation of NPs is increased in cancer due to inadequate lymphatic drainage, which enables the nanocarriers to transfer their payloads to tumor cells. These procedures result in the EPR effect, which is one of the drivers behind the passive targeting approach [34]^[24]. In conjunction with the EPR effect, the tumor milieu plays a significant role in the passive distribution of nanomedicines. One of the metabolic traits of cancer cells is glycolysis, which serves as the primary energy supply for the development of cancer cells ^[16]. The tumor microenvironment’s pH is decreased by glycolysis, which creates an acidic setting. As a result, some pH-sensitive NPs are activated by the lower pH and can release medications close to cancer cells [35]^[25]. Topotecan (TOPO) was encased in mesoporous silica nanoparticles (MSNs), and the nanosystem allowed the drug’s active form to be delivered in endosomes/lysosomes (pH 5.5) upon the internalization of nanoparticles. A pH-sensitive coating, a multimodal gelatin shell that protected TOPO from hydrolysis and premature release, and several anchorage sites for marking targeted ligands for preferential uptake in tumor cells were the hallmarks of MSNs. The nanosystems effectively destroy tumor cells while not affecting normal cells’ survival. On the other hand, free TOPO could not kill both cell lines due to the drug’s deactivation. This revolutionary nanodevice represents a step ahead in developing new cancer-fighting weaponry [36]^[26]. A topotecan-entrapped liposomal nanoformulation (LNP) was developed based on a loading process that entails the production of a copper water-soluble camptothecin complex. The same loading process developed for irinotecan was followed to produce an LNP topotecan formulation (Topophore C). At a final drug-to-lipid (D/L) mole ratio of 0.1, the entrapment efficiency of topotecan was noted to be >98%. Greater D/L ratios were possible; however, in vitro drug release tests revealed that the ensuing formulations were less stable. Topotecan plasma half-life and AUC were raised 10- to 22-fold in mice after Topophore C treatment, compared to free topotecan. Topophore C was noted to be 2-to 3-fold more toxic than free topotecan, but it had considerably superior anti-tumor effectiveness with no adverse effects. Based on the inferred findings, it can be inferred that Topophore C is a promising pharmacological candidate for treating platinum-resistant ovarian cancer [37]^[27]. Topotecan would continue to benefit from the targeted site delivery by utilizing nanocarriers. Anti-epidermal growth factor receptor (EGFR) and anti-human epidermal growth receptor 2 (HER2)-immunoliposome formulations substantially boosted topotecan internalization compared to the non-targeted counterparts and free topotecan, resulting in enhanced cytotoxic activity and superior antitumor efficacy against HER2-overexpressing human breast cancer (BT474) xenografts. Topotecan’s targeting capability and pharmacokinetic properties were considerably improved when it was stabilized in nanoliposomes, enabling potent and effective formulations against solid tumors [38]^[28]. Topotecan was further reported to have enhanced efficacy in ovarian cancer. An appreciable particle size and entrapment efficiency of 60.9 ± 2.2% were obtained in a nanometric range. The formulated nanoparticles illustrated a sustained release in physiological and acidic tumor microenvironmental conditions. The nanometric size enabled ideal internalization in SKOV3 (ovarian cancer) cell lines over time compared to topotecan in a soluble form, with a 13.05-fold rise in bioavailability when evaluated for pharmacokinetic attributes [39]^[29]. The polylactic-co-glycolic acid (PLGA) nanocarrier was also used to formulate topotecan (TPT), which improved the drug’s efficacy by reducing the accelerated conversion of the bioactive lactone form to the inactive carboxylate form. TPT’s stability was ensured by maintaining the drug-containing phase at an acidic pH. The drug maintained its active lactone form by lowering the pH of the inside of nanoparticles, which led to a 15-day biphasic release profile. Furthermore, compared to a neat drug, the cytotoxicity screening and in vivo antitumor effectiveness revealed considerable potential for greater proliferation inhibition ^[14]. Nanostructured lipid carriers (NLC) incorporating topotecan (TPT-NLC) were fabricated in hydrogels with hydroxyethyl cellulose and chitosan (TPT-NLC-HEC and TPT-NLC-Ch). For around 30 days, the said formulations retained the drug and nanoparticle dispersions stably. TPT release was dramatically reduced when nanoparticles were added to gels. TPT-NLC-HEC boosted permeability by 2.37 times compared to TPT-HEC (11.9 and 5.0 g/cm², respectively). Nanoencapsulation significantly increased TPT cytotoxicity when analyzed in B16F10 melanoma cells. With an IC50 value of 5.74 g/mL, TPT-NLC was noted to be more toxic than free TPT, whereas free TPT had an IC50 of >20 g/mL. Because the skin penetrated values of TPT from the established formulation (TPT-NLC) were higher than the melanoma IC50, it may be stated that chemotherapeutic permeated quantities may be adequate for a therapeutic impact [40]^[30]. TPT-SLNs were integrated into a thermoresponsive hydrogel system (TRHS) to create TPT-SLNs-TRHS, which allowed for controlled drug release and reduced drug-associated toxicity. When TPT-SLNs-TRHS was injected into the rat’s rectum, it showed good gelation capabilities. Furthermore, drug release was demonstrated to be controlled over an extended period for the integrated TPT. TPT bioavailability was improved with enhanced plasma concentration and area under the curve (AUC) in pharmacokinetic investigations. Furthermore, compared to the test formulations, it significantly improved antitumor impact in tumor-bearing animals. The study inferred that SLNs combined with TRHS could be a potential source of antitumor drug delivery with improved control over drug release, with no associated toxicity [41]^[31]. To augment topotecan’s transport to the lymphatic system, a primary conduit for cancer metastasis, and further enhance topotecan’s bioavailability and retention in target organs such as lung and brain, a research group formulated topotecan-loaded polymeric nanoparticles [42]^[32]. The cumulative percentage of topotecan release from the nanoformulation after a time period of 120 h were 91.56 and 92.02%, respectively, according to the results of in vitro release assays for the nanoformulation and free drug as a reference standard. PLGA nanoparticles with topotecan loading displayed a protracted release pattern in the studied time frame. Following 6 h after treatment, topotecan distribution was noted to be larger in each target organ after administration of the topotecan nanoformulation at a dose of 4 mg/kg than following delivery of the free drug. A similar pattern was seen following oral administration. The substantial intensity of luminescence was demonstrated for six hours following the injection of the nanoformulation. Higher luminescence in lymphoid tissues was noted, which was coherent with the quantitative observation of significant topotecan in the said tissues after intravenous administration of these nanoparticles. The outcomes of this study imply that topotecan NPs may produce superior therapeutic outcomes because they have a better pharmacokinetic profile and are efficient enough to distribute the drug more effectively to lymphoid tissues, the lung, and the brain as contrasted to the free drug [42]^[32].

3. Active Targeting

When the nanocarriers reach the tumoral zone, they face a challenging situation. Tumoral aggregates comprise various cell types, ranging from tumoral cells to immunological, supporting, and healthy cells from the extracellular matrix [47]^[33]. Consequently, nanocarriers must be able to distinguish malignant cells and localize their action on them to accomplish an effective therapeutic effect. By binding targeting moieties to the particle surface, this capability can effectively be included in the nanocarriers [48]^[34]. These targeting components are small compounds or macromolecules that engage specific receptors on tumor cells’ surfaces. These cellular receptors are found in healthy cells in many cases, such as for the extensively used targeting moieties folic acid [49]^[35], transferrin [38]^[28], and sugars [50]^[36]. However, because tumor cells have a larger nutrition need, their population is much higher than healthy cells. As a result, this receptor overexpression can be leveraged to selectively deliver therapeutic medications to tumor cells. Another option is to produce synthetic targeting components that are more selective and efficacious at binding to specific receptors [51]^[37]. Mesoporous silica nanoparticles laden with topotecan were synthesized and then surface-conjugated with folic acid (FTMN) to increase the drug’s effectiveness in treating retinoblastoma (RB) cancers. In physiological settings, the particles were nanosized and showed a controlled release of the entrapped drug. Compared to non-targeted nanoparticles, the folic acid-conjugated nanoformulations had a phenomenal absorption in RB cells. These findings strongly suggest that cellular uptake was regulated by receptor-mediated endocytosis. Compared to other formulations, FTMN had a considerably larger cytotoxic effect in Y79 cancer cells due to its higher cellular absorption. FTMN successfully triggered cancer cell death with a 58% effectiveness. The anticancer efficiency of TPT nanoformulation in Y79 cancer cells was superior to that of native drugs or unconjugated nanoparticles, according to the findings. FTMN revealed a decrease in overall tumor volume compared to the other group and fewer tumor cells in histological staining. Consequently, a folic acid-conjugated nanocarrier system could be a promising therapy option for RB [52]^[38]. Transferrin-decorated multifunctional nanoparticles (NPs) based on γ-cyclodextrin for tumor-targeted therapy were reported. The formulated NPs were found to cause a considerable increase in cellular uptake in MDA-MB-231 tumor cells leading to cell death. The transferrin-targeted NPs were proven effective carriers of TPT in vivo experiments using an MDA-MB-231 tumor xenografted mice model. TPT has the preferential ability to deliver chemotherapeutics to Tf receptor-positive MDA-MB-231 tumor cells, increasing drug uptake into the tumor cells and intensifying their toxicity [53]^[39]. Irrespective of the availability of numerous nanocarriers, researchers worldwide have explored liposomes as a suitable carrier system for the targeted delivery of topotecan employing specific ligands.

4. Combinatorial Drug Therapy Employing Topotecan

One of the cornerstones of cancer therapy is combinatorial therapy, which utilizes two or more chemotherapeutics to target cancer-inducing or cell-sustaining mechanisms [56]^[40] selectively. Even though the mono-therapy technique is still a prevalent type of cancer treatment, it is typically thought to be less efficient than the combination therapy strategy. Typical mono-therapeutic approaches non-selectively target actively multiplying cells, inevitably resulting in the death of both malignant and healthy cells. Chemotherapy can harm the patient and comes with several hazards and side effects. It often leads to drug resistance and cancer cell survival (Figure 4). It can also significantly lower the patient’s immune system, by weakening bone marrow cells and making them more vulnerable to host illnesses. The toxicity aspect of combinatorial therapy is greatly reduced because diverse channels will be targeted, even if it can still be harmful if one of the medications is a chemotherapeutic. Combinatorial therapy has a synergistic effect, necessitating a reduced therapeutic dosage of each chemotherapeutic separately [57]^[41]. Combinatorial therapy may also provide cytotoxic effects on cancer cells while preventing harmful effects on healthy cells. In a drug-sensitive cancer cell, a strong drug-target interaction occurs, followed by the internalization of the anticancer drug at the target site leading to cell death. In contrast, in a drug-resistant cancer cell, a weak/inactive drug-target interaction occurs, followed by the poor internalization of the anticancer drug at the target site leading to cell survival. Due to limited drug diffusion across the blood–brain barrier (BBB), the prevalence of MDR, and inadequate uptake into tumor tissues, chemotherapy for brain malignancies continues to be a challenge. Tamoxifen was integrated into the liposomes, and wheat germ agglutinin was coupled to the surface of liposomes. Topotecan was further loaded into the preformulated liposomes. The ligand-modified topotecan liposomes displayed a considerable inhibitory effect in the MTT experiment compared to the unconjugated formulation, implying that both the ligands confer robust drug delivery benefits into brain tumor cells following immediate drug exposure. The lowering of C6 glioma tumor spheroid volume and apoptosis was also noted. The combinatorial effects were observed in brain tumor-bearing rats, culminating in a considerable enhancement in the overall survival of the treated rats. Furthermore, data from an extended treatment group showed that survival could be improved, implying that protracted chemotherapy with topotecan liposomes modified with TAM and WGA would be favorable for the efficacious treatment. To summarize, targeted topotecan liposomes enhance topotecan trafficking across the BBB, demonstrating dual-targeting benefits. These results could pave the way for more noninvasive brain tumor treatments [59]^[43]. A combinatorial therapy involving paclitaxel (Pac) and topotecan (Top) in a Pac-Top ratio of 20:1 w/w was incorporated into folate-anchored PEGylated liposomes (FPL-Pac-Top) for effective ovarian cancer treatment was reported by a group of researchers. Toxicity to blood cells was found to be minimal in hematological experiments. Long circulatory behavior and preferential accumulation of FPL-Pac-Top in the ovaries were observed in vivo. In addition, FPL-Pac-Top revealed lower necrosis and greater apoptosis. Compared to the Pac-Top solution, Kaplan–Meier survival analysis demonstrated a two-fold increase in the survival time by FPL-Pac-Top. The potential efficacy of FPL-Pac-Top was related to some characteristics, including thermosensitivity, extended circulatory nature, and targetability. Such an envisaged method could be a revolutionary chemotherapeutic technique for safe and effective cancer-targeting options [60]^[44]. To increase the penetration of topotecan through the gut and breast cells to treat breast cancer, tamoxifen citrate was added to the formulation to generate dual drug-loaded nanoparticles [62]^[45]. Tamoxifen is a P-glycoprotein (P-gp) inhibitor that helps decrease the drug’s adverse effects by lowering its dose. It was observed that the optimized dual drug-loaded nanoparticles had a smooth and spherical architecture. The in vitro release study demonstrated the sustained release of both the entrapped drugs. The ex vivo gut permeation investigation showed that TAM improved TOP’s penetration and raised its bioavailability by 1.9-fold. Cell line experiments on MCF-7 cells were conducted further to establish the penetration and cytotoxicity of the drug combination. Comparing the dual drug-loaded nanocarrier system to native drugs alone and in combination, it was found to be significantly more lethal at low concentrations. The P-gp inhibition caused by tamoxifen on the cell surface and the nanoparticles’ relatively small size, making it easier for them to enter cells, are most likely responsible for the said effect. Additionally, compared to pure drugs or drug mixtures, dual drug-loaded nanoparticles demonstrated lower IC50 values leading to superior cell death. The reason could be that tamoxifen inhibits P-gp; it helps restrict topotecan from effluxing off breast cancer cells (MCF-7). The obtained results implied the superior cytotoxic potential of the system in facilitating breast cancer. Hence, it is imperative to state that combinatorial therapy was more efficacious in treating breast cancer than single therapy.