3.1.1. Non-Melanoma Skin Cancer and Cutaneous Melanoma
The most common skin cancers are the non-melanoma types, i.e., basal cell carcinoma (BCC), and squamous cell carcinoma (SCC), which are derived from epidermal keratinocytes and are frequently detected in an early resectable stage [
43]. In 2018, the incidence of non-melanoma skin cancer rose to 1,042,056 cases, corresponding to 5.8% of all cancers in the world, with a particularly increased incidence in Australia and New Zealand [
1]. Primarily, those types of skin cancers are usually removed by surgery or desiccation, if possible, or otherwise with radiation therapy. Additionally, PDT has been successfully applied in superficial skin cancers, using photosensitizers such as aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) [
43]. Still, one major problem concerning this approach is that those tumors occur mainly in chronically sun-exposed areas, such as the face, ears, scalp, and hands; thus, both the physical and emotional impact of excision are significant for patients [
44]. Furthermore, the application of photosensitizers in these cases is limited by the low penetration into the tumor tissue due to their poor solubility, in addition to the related phototoxicity, which can last up to several weeks after PDT [
43,
45].
Within the nanotheranostic field, research is focused on rationally combining imaging and therapeutic modalities, which restrict the treatment locally in the tumor area, while improving the penetration of the drugs and/or photosensitizers. Recently, a “bottom-up” method was developed to overcome these limitations of PDT. Briefly, this strategy involved the nano-assembling of the photosensitizer phthalocyanine and the anticancer drug mitoxantrone, associating both optical and chemotherapeutic actions [
45]. In addition, the nanosystem was assessed for its capability of converting light into heat; indeed, a mild temperature elevation was found, but should be carefully explored to be further proposed for a PTT effect. As a first attempt, the targeting action of this system was verified in breast cancer MCF7 cells and colon SW620 cancer cells, which suggested that this strategy can be potentially suitable for treatment of different tumors, such as skin cancer. Another promising characteristic of the nanoparticles was the fast and significant accumulation in the tumor tissue. Although the nanoparticles also spread quickly to other organs (e.g., the liver and lungs) during the first hours (1–2 h), those were rapidly eliminated from the body within 24 h. Overall, this supermolecular nanoplatform was able to accumulate in tumors and emit fluorescence imaging, as well as inhibit cancer growth by a synergistic effect with laser irradiation and chemotherapy [
45].
A study combined both PTT and imaging actions with a biocompatible 9 nm nanotheranostic platform made of gadolinium and copper chalcogenides, with a strong absorbance in the NIR range [
46]. Bovine serum albumin was used as a biocompatible mediator and as an agent to improve photostability. This nanotheranostic allowed a bimodal imaging-guided PTT, responding to 980 nm laser irradiation for 5 min, with temperatures increasing up to 50 °C at the tumor site, and demonstrated inhibitory effects on tumor growth [
46]. In terms of its biodistribution, this nanosystem accumulated mainly in organs of the reticuloendothelial system (RES) (e.g., liver and spleen). An ovarian carcinoma cell line (SK-OV-3) was selected to assess the efficacy of this system, rather than the skin cancer model; nevertheless, due to the broad and flexible modalities, we considered that this technology could be also translated into a therapy for skin cancer.
Among the skin cancers, cutaneous melanoma (or melanoma of the skin) is the most common type of melanoma, arising from complex genetic mutations in melanocytes [
47]. The tumor microenvironment in melanoma is also very heterogeneous, with complex vascular networks and immunogenicity, as well as induced acquired resistance to treatments by upregulation of multidrug resistance (MDR) mechanisms [
48]. Treatment of cutaneous melanoma has improved over the last several decades, but the survival of patients with advanced disease is still poor [
48,
49]. The number of melanoma cases has increased over the last years, with an estimated incidence of 287,723 cases in 2018, which corresponds to 1.6% of all cancers [
1]. In comparison with non-melanoma skin cancer, which is responsible for 65,155 deaths (0.7%) per year, melanoma mortality reaches 0.6% (60,712 deaths) despite being less frequent [
1]. Conventional therapies (e.g., dacarbazine) for melanoma present many limitations, such as reduced target specificity, severe adverse effects, and eventually MDR. Recent targeted therapies with kinase inhibitors to suppress the MAPK pathway downstream, specific for BRAF V600 mutation (e.g., vemurafenib and dabrafenib) or MEK inhibitors (e.g., cobimetinib and trametinib) are currently available for those patients with advanced melanoma. However, those novel treatments cause cutaneous toxicities and show a short-term response due to acquired resistance. On the other side, immunotherapy (e.g., ipilimumab and nivolumab) present also a limited response due to acquired resistance and immune-related side effects [
50].
In parallel to the development of more potent and specific therapies, an important step for the success of melanoma treatment is its early detection [
51]. When there are no metastases, this cancer can be removed by surgery; however, the risks of intervention and of cancer recurrence must be measured in patients with locally advanced tumors (Stages I–III). In those situations, treatment with high doses of interferon-α is administered to improve chances of disease-free and overall survival. As a new line of research, adjuvant treatments are being explored, comprising immune and target therapies in combination with chemotherapy, in addition to neoadjuvant strategies, applied before surgical removal of the tumor [
52,
53]. In this setting, nanotheranostics might have great involvement, if capable of reducing the size of the tumor and facilitating an effective surgery, or even avoiding it by complete tumor elimination. Moreover, theranostic nanoparticles can be useful for localizing the tumor at an early stage through imaging techniques or for monitoring the treatment delivery directly to the tumor cells [
54].
In recent studies, both tumor necrosis factor (TNF) or epidermal growth factor receptor (EGFR) have been selected for targeting treatment approach, as well as the signal transducer and activator of transcription 3 (STAT3), to enhance the apoptosis of melanoma cells [
55]. As a first approach, Labala et al. developed a layer-by-layer chitosan-coated gold nanoparticles for local delivery of imatinib to melanoma cells using STAT3 siRNA as the targeting moiety for the inhibition of melanoma cell growth [
56]. These nanoparticles with a size around 150 nm were able to reduce tumor growth in a concentration-dependent way, reducing the protein expression by almost 50% in murine melanoma cell models [
56]. Thus, those gold nanoparticles provided the ideal optical imaging and tracking functionalities to produce a theranostic platform.
As previously mentioned, gold nanoparticles have been extensively used for both diagnosis and treatment modalities in melanoma cancer, ideally at an early phase and allowing for localized distribution [
55]. Gold nanoparticles are very versatile and can be bioconjugated with biomolecules or drugs, which make them suitable for dealing with distinct molecular pathways, such as those involved in heterogeneous cancers, including melanoma [
43]. In the field of nanotheranostics, gold nanosystems can be applied for imaging, detecting circulating tumor cells, and, mostly, for a targeted drug delivery, increasing patients’ overall survival [
43]. Previously, our research group designed hybrid gold nanoparticles for a photothermal strategy in cutaneous melanoma targeted therapy, which demonstrated potential to destroy melanoma cells at their initial stage by photoactivation and thermoablation, considering a neoadjuvant setting [
18]. Nanoparticles were successfully coated with hyaluronic and oleic acids (HAOA) and conjugated with epidermal growth factor (EGF) as a key relevant medical peptide. HAOA-coated gold nanoparticles show a broad absorbance band instead of a narrow absorbance peak (lower activation energy) around 700 nm, allowing an adequate relation between low activation energy and high depth, which enables the laser to irradiate this superficial cancer completely [
18]. In vivo assays showed that those spherical gold nanoparticles were actively internalized by tumor cells through EGFR-mediated endocytosis. Furthermore, the nanoparticles associated with NIR-based photothermal therapy (808 nm laser irradiation for 5 min) reduced tumor volume by 80% and caused several coagulative necrotic foci on tumor tissue, but no significant damage of the surrounding tissue or any other side effects [
18]. Additionally, the same gold nanoparticles were directly coupled with another cytotoxic abietane (i.e., 6,7-dehydroroyleanone) [
57], and their design and composition were adapted for polymeric nanoparticles loaded with a novel anticancer drug (i.e., parvifloron D), demonstrating also promising results in targeting and reducing the growth of melanoma cells [
19]. In summary, our studies allowed us to conclude about the flexibility and adaptability of these systems, in addition to applications such as the conjugation of a photosensitizer, evolving to a triple-action nanotheranostic system.
Trying another approach with PTT, Chen et al. (2018) employed an NIR-absorbing polymer such as sulfonated poly(
N-phenylglycine), and a supercharged green fluorescent protein (ScGFP) to form a hybrid nanoparticle with imaging-guided therapeutic action [
58]. These novel theranostic nanoparticles were able to convert NIR light (808 nm laser irradiation) into local ablation of melanoma cells after 5 min, without damage to the surrounding tissues [
58].
As a conclusion, and considering the enormous advantages made in this field, especially in light-triggered systems for PTT, it is possible that soon enough skin cancers, including melanoma of the skin, can be treated with a safe and efficient image-guided and laser-based treatment, focusing on the tumor area and, eventually, eliminating the tumor without the need of surgery.
3.1.2. Head and Neck Cancers
Head and neck cancers represent approximately 4% of all diagnosed cancers [
1,
59]. Around 90% of these tumors are squamous cell carcinomas, which include tumors of the oral cavity, larynx, pharynx, and glands [
1,
60]. The incidence of these tumors is approximately 550,000 cases per year worldwide [
60,
61]. Moreover, according to recent estimates, the burden of head and neck squamous cell carcinoma (HNSCC) is increasing in low income countries, such as those in the Asia-Pacific region and Africa [
59,
60]. The most relevant risk factors are tobacco smoking, alcohol consumption, and human papillomavirus (HPV) infections [
61]. Multidisciplinary standard treatment for these locally advanced tumors includes surgery, postoperative radiotherapy, and/or chemoradiotherapy [
62]. Tumor location defines the treatment selection, with tumors in the oral cavity usually being resected by surgery, and oropharynx, nasopharynx, hypopharynx, and laryngeal carcinomas treated with radiation and chemotherapy to preserve the organ structure. Nevertheless, morbidity associated with invasive procedures, incomplete resection, and drug resistance reveals an unmet need for less invasive and safer therapeutics.
Regarding molecular pathways, HNSCC shows an 80–90% overexpression of EGFR, considered as an implicated key target for chemotherapies [
63,
64]. Associated with EGFR is the signal transducer and activator of transcription 3 (STAT3), constitutively activated in several cancers including HNSCC, and this contributes to uncontrolled tumor growth (via anti-apoptotic mechanism) [
65]. As most patients do not respond or develop resistance to current treatments [
61], nano-delivery systems can allow higher drug bioavailability, local targeting action, and reduced toxicity to healthy tissues [
66,
67]. Hybrid nanoparticles made of polymeric, metallic, and other bioactive compounds and functionalized with targeting moieties are being studied to mitigate these limitations.
First described in 2011, AGuIX
® products are ultrasmall (size < 5 nm) paramagnectic gadolinium-based nanoparticles with a polysiloxane matrix, providing both contrast agent properties and radiosensitizing efficacy. These nanosystems have been developed to improve MRI diagnosis of solid tumors while providing treatment with radiotherapy and are currently under translation to the clinics [
68]. The main advantages of these nanostructures are the high tolerance (few side effects) and renal elimination, as well as the possibility of using different administration routes (intravenous or intratumoral injection). Furthermore, this nanosystem allows for the combination of photosensitizers (e.g., tetraphenylporphyrin derivative) for another therapeutic action throughout PDT. Thus, this nanotheranostic offers a dual optimized therapeutic effect by radiation therapy and light-based photosensitizing modality, which can benefit from MRI properties in the detection of the tumor location and guide the treatment within its borders [
68].
In this context, it seems that a new generation of nanotheranostic particles is under study, such as those comprising new metallic materials (with different properties) and surface functionalization with multiple targeting moieties. Among all available nanostructures for light-based applications, gold nanoparticles have been widely studied due to their high absorption coefficient, potential versatility, and functionalization [
69]. Recently, a dual theranostic system made of gold nanoparticles was explored to target the activation of STAT3 in head and neck cancer, using a combination of cell-surface nucleolin and radiosensitizing approaches [
70]. Nanoparticles were functionalized with the targeting oligonucleotide and were radiolabeled to enhance cancer cell uptake. Internalization of the oligonucleotide gold nanoparticles induced apoptosis via enhanced DNA double-strand break formation. Eventually, these nanosystems can be applied as a primary option as an adjuvant radiation therapy for post-surgery or non-resectable head and neck cancer [
70].
Moreover, 20 nm spherical gold nanoparticles coated with glucose and cisplatin were produced, in combination with radiation treatment, to ensure an imaging guided treatment for head and neck cancer [
71,
72].
d-glucose was previously described as a natural contrast agent for CT scan, MRI, PET, and SPECT [
73]. In the work conducted by Davidi and Dreifuss, glucose worked as a radiosensitizer and as a carrier to deliver cisplatin by means of glucose transporter-1 receptor internalization, which was overexpressed in head and neck cancer cell lines (e.g., A431) [
71,
72]. To guarantee the activity of glucose as a targeting ligand and improve the receptor mediated endocytosis, conjugation with the gold nanoparticles was conducted with glucose’s second carbon atom. Here, a synergistic result was observed in terms of tumor reduction when using the conjugated gold nanoparticles and cisplatin, since this metal can also act as a radiosensitizer. Nevertheless, it was found that, after injection in tumor-bearing mice, the conjugated nanoparticles not only were present in the tumor site and kidneys but also managed to cross the blood–brain barrier (BBB) and reach the brain tissue (about 5.8%) [
71,
72]. Identical to other approaches, these gold nanoparticles were coated with a natural ligand that fulfills a specific interest in anticancer treatment and potentially improves tumor reduction, compared to the conventional available treatment. However, the effects of the undesired and unspecific accumulation of the therapeutic agents and nanocarriers should be carefully measured fostered by a safer medical treatment.
In addition to polymeric-based hybrid nanoparticles, multifunctional systems can also comprise lipid molecules associated with any inorganic material. As an example, porphyrin (inorganic PTT agent) has been self-assembled with lipid film, made of pyropheophorbide-lipids, cholesterol, and polyetheneglycol (PEG2000-DSPE), forming a nanotheranostic agent called porphysome [
74]. This system combines a tracking modality based on PAI and fluorescence imaging allied with PTT treatment (
Figure 4). Each imaging technique has a different aim, since the photoacoustic signal can detect the intact nanoparticles present at the tumor site, and the fluorescence imaging can track the delivery of the porphysome. Both features allowed a real-time guidance and dose adjustment of the treatment [
74]. In this study, the porphysome showed a tumor-specific accumulation (highest peak after 24 h post-injection) and a general biodistribution to the spleen, liver, and small intestine of a rabbit model with buccal carcinoma. Nonetheless, these nanosystems were able to clearly delineate the tumor margins (a five-fold increased signal) and promote tumor necrosis after irradiation for 100 s (around 1.6 min) at 671 nm. No cellular damage nor any other side effects were observed in other organs (including the heart, liver, spleen, lung, kidney, and salivary gland) [
74].
In summary, photoacoustic and other imaging-guided modalities are under assessment to deliver a more precise, efficient, and safe treatment for head and neck cancers. Indeed, PAI have been recognized as an early procedure for starting radiation therapy in patients with head and neck cancer [
75] and, thus, providing a real-time imaging of dynamic changes in tumor oxygenation, which influences the necessary dose and the overall efficacy of the radiation treatment. Additionally, the combination of PTT and PAI might promote a synergistic therapeutic effect since the imaging modality also allows optical excitation in the NIR range, promoting a local heating of the tumor tissue [
75]. Although clinical use of NIR fluorescence-guided surgery is limited due to a few approved contrast agents, further development of specific fluorescent probes, such as anti-EGFR antibodies, can increase the use of these techniques for diagnosis, treatment, and the follow-up of head and neck cancers [
39]. Nanotheranostics can present specific characteristics that confer the specificity needed to treat a subpopulation of tumors; they otherwise might be suitable for many different tumors such as those of breast, prostate, or thyroid cancer. NBTXR3
® is an example of a promising nanotheranostic made of hafnium oxide nanoparticles combined with radiation therapy that was first assessed for head and neck cancer and is currently being assessed in clinical trials for other solid cancers, such as rectal cancer, prostate cancer, and breast cancer (
Table 1).
3.1.3. Thyroid Cancer
Thyroid cancer accounts for 1% of all cancers and is the most common endocrine tumor, with 64,300 cases and 1980 deaths in the USA in 2016 [
76,
77]. The incidence has increased over the past few years, reaching 567,000 cases worldwide in 2018 [
1]. Furthermore, this cancer is responsible for 5.1% of the total estimated female cancer burden, with a global incidence three times higher in woman than in men [
1]. The survival rate is still quite high, leading to 0.4% of deaths [
1,
77,
78]. This cancer also presents a complex etiology, which is still not well understood, and is associated with multiple risk factors, among which is childhood neck radiation, Hashimoto’s thyroiditis, family history of thyroid adenoma or cancer, familial adenomatous polyposis, obesity, smoking, and hormonal exposures [
1,
76]. Overall, thyroid cancer arises from follicular cells in the thyroid and is the most common endocrine malignancy [
79]. Advances in nuclear technologies and new diagnostic techniques allowed one to improve the success of differentiated diagnose of thyroid cancer, which also led to the increased incidence of this cancer in many countries [
1]. Along with these technological contributions, theranostic radioiodine has also emerged for a personalized management based on molecular imaging (used since 1950) and highly effective treatment [
79].
In this context, nanotheranostics are gaining space in recent years for applications in optimization of a tailored thyroid cancer medicine. Up until now, several studies reported the use of nanoparticles in the diagnosis and/or treatment of thyroid cancer. Carbon nanoparticles, for example, are widely investigated in this type of tumor [
80,
81,
82,
83]. Wang and collaborators evaluated the efficacy of carbon nanoparticles of about 150 nm to be accurately identified in patients undergoing central lymph node dissection surgery [
82]. The study aimed at reducing an erroneous excision, as well as evaluating the value of this nanosystem in obtaining a faster recovery of the parathyroid gland function. In fact, the authors concluded that it was possible to delimit the lymph nodes with high precision, therefore allowing the surgery to be as accurate as possible, so that other glands, such as the parathyroid glands, were not affected [
82].
Another study evaluated the efficacy of perfluorocarbon nanoparticles as a diagnostic method for thyroid cancer, by ultrasound molecular imaging, and for SHP2-targeted cancer treatment [
84]. The nanoparticles were produced by double emulsion, in which trichloromethane was first dissolved in poly(lactic-co-glycolic acid) (PLGA), followed by the addition of perfluorocarbon. The obtained polymeric precipitate was further activated by the addition of carbodiimide crosslinker using EDC/NHS coupling, and the SHP2 antibody solution and polyethylenimine were then added. Finally, DOTA-NHS was conjugated to the nanosystem for biomedical imaging purposes. Results showed that the formulation had a high affinity for thyroid cancer cells due to the overexpression of SHP2 in these tissues compared to healthy thyroid tissue, and could be activated by low-intensity focused ultrasound (LIFU)-triggered radiation to enhance ultrasound molecular imaging, allowing for the identification of the tumor [
84].
One study developed silicon dioxide nanoparticles conjugated with a specific ligand for thyroid cancer, the thyroid-stimulating hormone receptor (TSHr), and loaded with a well-known anticancer drug (e.g., doxorubicin) [
85]. As previously described, the tumor environment, in which the bond between doxorubicin and cis-aconitic anhydride is broken and the cytotoxic compound can be rapidly released, shows an acidic pH. Therefore, for the production of these nanosystems, doxorubicin was first conjugated with cis-aconitic anhydride, followed by conjugation with PEGylated silicon dioxide nanoparticles [
85]. Briefly, the nanoparticles were obtained by SiO2 junction with NH
2 groups exposed with succinimidyl carboxyl methyl ester (mPEG-NHS) and orthopyridyl disulfide PEG succinimidyl ester (PDP-PEG-NHS). To ensure the specificity of the system, TSH was bound to these disulfide bond pathways. In vitro studies in thyroid cancer cells (FTC-133 cell line) demonstrated a significant increase in toxicity, approximately 7.3 times stronger with this nanoformulation compared to the free drug. Indeed, the IC
50 value of free doxorubicin was 2.32 μM, whereas that of this formulation was only 0.32 μM. Another strong factor highlighted by the authors was the decrease in cardiotoxicity effects by the nanosystem compared to the free doxorubicin [
85].
As an alternative for polymeric nanoparticles, Sun et al. explored the advantages of polymeric photothermal agents instead of carbon- and metal-based compounds for formulation of nanotheranostics, and these advantages include improved optical absorption, photostability, and biocompatibility [
86]. In this study, nanoparticles made of narrow band gap D−A conjugated polymer (TBDOPV−DT) were produced for PAI and photothermal therapy, triggered by an NIR laser radiation at 1064 nm (increased tissue penetration capacity). In vitro testing demonstrated that the nanoparticles were efficiently taken up by HepG2 and HeLa cell models, while reducing their viability if treated with combined laser irradiation and TBDOPV−DT nanoparticles. In terms of biodistribution, the nanoparticles showed an early accumulation in both liver and lungs. As expected, the nanosystem showed strong photoacoustic signals, which was also beneficial for photothermal performance (photothermal conversion efficiency of 50%) and thus could completely inhibit the growth of tumors and avoid recurrence within 20 days [
86]. Overall, nanotheranostics may unravel new approaches for diagnostic and treatment of different clinical and histological features in thyroid cancer, helping to clarify underlying pathogenesis that are still unclear in some of these tumors.
3.1.4. Breast Cancer
Breast cancer is the second most common cancer in the world and the most frequent in women [
1,
87]. In the USA, for instance, among females, the mortality rate of breast cancer is only surpassed by the lung cancer mortality rate, it is estimated that one in eight women will develop invasive breast cancer [
1,
87]. In 2018, more than 330,000 new cases of invasive and non-invasive breast cancer are expected in women [
88]. Despite the scientific development that allowed advances in the treatment and detection of the disease in earlier states, more than 40,000 women due to breast cancer are expected to die in 2018 in the USA [
87,
88]. In men, the number is much lower; however, about 2500 new cases of invasive breast cancer are expected [
88].
Breast cancer is a complex and heterogeneous type of cancer, characterized by the occurrence of multiple molecular alterations, which can be used as diagnostic and prognostic markers of the disease [
87,
88]. The most common cause of hereditary breast cancer is an inherited mutation in the BRCA1 or BRCA2 gene, which are tumor suppressor genes involved in such essential functions as DNA repair [
87,
88]. There are various biological processes and genetic mutations that lead to the appearance of breast cancer and sensitivity to various drugs, such as hormone receptor, human epidermal growth factor receptor 2 (HER2), EGF, vascular endothelial growth factor (VEGF), mechanistic target of rapamycin (mTOR), and cyclin-dependent kinase 4/6 (CDK4/6) [
89]. Women, compared to men, present a 100-fold increased risk of having breast cancer; furthermore, aging and heredity also contribute as predisposing factors to the disease [
88]. Many non-genetic risk factors of breast cancer are involved, such as race and ethnicity, benign breast conditions, proliferative breast lesions, lobular carcinoma in situ or lobular neoplasia, chest radiation therapy, exposure to diethylstilbestrol, lifestyle and personal behavior-related risk factors of breast cancer, birth control and contraceptives, hormone replacement therapy after menopause, excessive alcohol consumption, significant overweight or obese, not having children or not breastfeeding, and a lack of physical activity [
88].
With the improvement in science and medicine, targeted therapies that increase the efficacy and precision of the treatment and decrease the toxicity are now an ambitious solution under development [
89]. Several studies have reported the use of nanoparticles as a strategy to combat breast cancer, such as a new VEGF-targeted nanocarrier, composed of magnetic nanoparticles coated with albumin that carry doxorubicin and in which it conjugates monoclonal antibodies to VEGF, which increases their specificity for the tumor [
90]. Additionally, due to its magnetic core, this system can still be used for diagnosis, and its detection is possible by MRI after i.v. injection. The nanoparticles were synthesized by thermal decomposition of iron (III) acetylacetonate in benzyl alcohol, coated with BSA (bovine serum albumin) and PEG and conjugated with anti-VEGF monoclonal antibodies [
90]. In this study, researchers optimized the nanoparticle size to less than 50 nm, as well as the compounds present in the coating, since these are two important factors for the nanoparticles to remain longer in the bloodstream. Regarding the coating, nanoparticles were pegylated to guarantee an efficient loading process with doxorubicin, to prevent interaction with the plasma proteins and to avoid their absorption by RES, and managed to obtain superior results compared to clinically approved formulations [
90].
In another study, β-lactoglobulin nanoparticles were conjugated with folic acid to allow receptor-mediated endocytosis present in cells, and loaded with doxorubicin, which has been shown to be effective against MCF-7 and MDA-MB-231 [
91]. First, a β-lactoglobulin solution was incubated with the previously prepared solution doxorubicin hydrochloride for 30 min, to which acetone was added at a constant rate until it became milky. A glutaraldehyde aqueous solution was then added, and the solution was allowed to stir for a few hours. Conjugation with folic acid was done by adding the nanoparticle suspension to a pre-prepared solution of folic acid followed by 1 h of stirring and purification by centrifugation [
91]. This formulation was able to retain doxorubicin allowing non-release of the cytotoxic compound into healthy cells as well as the bloodstream, the release being made at the tumor site in response to the more acidic pH characteristic of the tumor environment. In vitro testing demonstrated that the nanosystem presented a greater toxicity profile compared to free doxorubicin after 72 h of incubation, against breast cancer cell lines [
91].
Another recent study was based on the grounds that miRNA-21 can be used as a diagnostic and therapeutic biomarker for breast cancer, so gold nanoparticles functionalized with a chemically modified miRNA-21 were developed [
92]. This method aimed at suppressing the function of miRNA-21 present in tumor tissues for the inhibition of cell growth and the death of apoptotic cells, while simultaneously using fluorophore-labeled DNA molecules hybridized with antimiRNA-21 for diagnostic purposes. Briefly, the nanoparticles were produced using the sodium citrate reduction method, followed by the addition of antimRNA-21 with labeled DNA molecules [
92]. Regarding metal-based systems for nanotheranostic approaches, spherical silver nanoparticles with fluroglucinol were produced according to a simple, green method [
93]. In short, silver nanoparticles were prepared by dissolving fluroglucinol in water followed by 20 min of stirring after which silver nitrate was added to the previous solution. Floroglucinol, a polyphenolic compound present in plants and marine algae, shows multiple therapeutic properties, e.g., antioxidant, anti-inflammatory, antimicrobial, anti-diabetic, anti-allergic, and antiretroviral. These nanosystems, which showed a small size (10–50 nm), were tested against MCF-7 breast cancer cell lines, demonstrating remarkable cytotoxic properties [
93].
Similar to gold and silver nanoparticles, Fe
3O
4 magnetic nanoparticles present broad optical absorption in the NIR range and increased possibilities for biopolymeric coatings and functionalization. In addition, magnetic nanoparticles have the advantage of potentiating supermagnetic proprieties that work as an MRI contrast agent [
94]. Hence, Fe
3O
4 magnetic nanoparticles conjugated with methotrexate were developed and further encapsulated in hepatitis B virus core (HBc) protein as a shield and stabilizer for the nanocarrier (
Figure 5) [
94].
This innovative system was tested in vitro for photothermal stability and irradiation-induced apoptosis, as well as in murine breast cancer 4T1 in BALB/c mice exposed to an 808 nm NIR laser for 5 min. Tumor size reduced in 10 days, comparable to treatment with nanoparticles alone or no treatment at all. MRI capability was also assessed both in vitro and in vivo, showing that this system could guide in the tumor position with great precision [
94].
Finally, a carboxyl-modified PEGylated poly skeleton, to which the herceptin antibody was modified to present a HER-2 specific surface, was added to superparamagnetic iron oxide nanoparticles (SPIONs), with perfluorohexane and paclitaxel [
95]. These SPIONs were activated by NIR laser and allowed the transformation of laser energy into thermal energy, causing the perfluorohexane to be vaporized and consequently release paclitaxel. Therefore, this nanotheranostic comprises another combination of photothermal therapy and chemotherapy for the treatment of breast cancer [
95].
In conclusion, multifunctional nanotheranostics including imaging and light-based therapies have been developed to improve the local action and reduction of the impact of chemotherapy in healthy breast tissue. This technique allows for the reduction of the size of the tumor, alike to melanoma and other operable solid cancers, working as a neoadjuvant therapy.
3.1.5. Prostate Cancer
Prostate cancer is one of the leading tumors in terms of incidence (7.1% of all cancers worldwide) and mortality (3.8% of deaths worldwide), surpassed only by lung and breast cancers [
1]. In recent years, both screening and diagnostic techniques for prostate cancer, as well as the clinical applicability of key molecular targets and receptors, such as the prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptors (GRPR), have considerably improved [
96,
97,
98]. PSMA is a type II transmembrane protein involved in many cellular functions, such as enzymatic functions, cell migration, survival, and proliferation [
97]. PSMA is overexpressed in almost all prostate cancers (90–95%), but is also present in healthy cells from the small intestine, proximal renal tubules, and lacrimal and salivary glands, which limits the dose used in radiation therapy [
96,
97]. Targeted therapies for PSMA have been studied in prostate cancer, mainly through antibodies and antibodies-drug conjugates [
96]. GRP receptors are overexpressed in prostate cancers and are found in the gastrointestinal tract [
98]. Interestingly, high upregulation of GRPR is specific to prostate carcinoma and occurs mostly during the early stages of this cancer [
99]. With regard to the relevant targeting molecules, the GRPR antagonists show increased advantages over the agonists, such as a favorable pharmacokinetics (longer receptor retention followed by rapid clearance), a higher affinity for those receptors and higher in vivo stability (e.g., 177Lu-NeoBOMB1, 68Ga-NeoBOMB1) [
98,
99]. In addition, to the targeting moieties, anticancer treatments such as taxanes (e.g., docetaxel and paclitaxel) have been explored in association with several imaging and treatment modalities (e.g., radiation therapy and ultrasound imaging) [
100,
101,
102]
Within this scenario, multimodal nanotheranostics could potentially improve sensitivity and specificity in the diagnosis of prostate cancer, for instance, by combining multiple targeting molecules for an effective treatment only in cancerous cells. Previously, the pre-clinical and clinical use of nanoparticles as potential theranostics, including metallic, polymeric, and lipid-based systems, have been reviewed elsewhere [
103]. In fact, research in this field has focused greatly on prostate cancer, aiming at an early and simultaneously diagnosis and treatment.
In a first approach to develop a combined theranostic nanocarrier, nanoemulsions and poly(
d,
l-lactide-co-glycolide) (PLGA) nanocapsules were produced, entrapping a photosensitizer molecule for PDT (chloro aluminum phtalocyanine) [
104]. This molecule was used as a therapeutic agent and for tumor localization by confocal laser scanning microscopy after fluorescence promoted by NIR laser irradiation at 670 nm [
104]. Both nanosystems presented a negative surface charge (approximately −40 mV) and a mean size around 200 nm, but nanocapsules were able to internalize the prostate cancer cells and promote phototoxicity more efficiently than nanoemulsions [
104]. Other research groups proposed a polymeric nanogel (<200 nm), showing intrinsic photoluminescence and encapsulating a naturally fluorescent anticancer drug (doxorubicin) [
105]; on the other hand, multi-layered PLGA nanoparticles (~200 nm) were produced for co-encapsulation of theranostic agents (e.g., curcumin, doxorubicin, hydrochloride, and indocyanine green), with chemotoxicity or phototoxicity profiles and intrinsic fluorescence, for a controlled pH-responsive release [
106]. Thus, in these two different approaches, it was demonstrated that multiple agents can be encapsulated into the same nanocarrier, or even the nanocarrier itself can be modified, to achieve both imaging and therapeutic actions.
In line with insights from PSMA-target therapies, researchers have also turned their attention to design PSMA-target theranostic nanocarriers for prostate cancer [
107,
108]. Folic acid is a natural PSMA-targeting ligand [
97] and has therefore been explored for functionalization of drug delivery systems. Flores et al. reported the use of folate-conjugated polymeric nanoparticles to deliver a cytotoxic peptide, CT20p, which reduces cancer cell migration and adhesion, consequently leading to the selective elimination of tumors, expressing chaperonin containing TCP-1 (CCT) [
107]. Results demonstrated that cell internalization could be mediated by overexpressed PSMA, due to the absence of folate receptors (PSMA (+) pre-treated PC3 and LNCaP cell lines) and that CT20p allowed for a selective elimination of prostate cancer cells, compared to doxorubicin alone [
107]. Another approach using also folate-functionalized nanosystems explored the use of MRI based on SPIONs, to increase the sensitivity and specificity of the diagnostic technique and reduce the toxicity associated with contrast agents [
109]. Hence, a norbornene-based magnetic copolymer was synthesized, comprising doxorubicin and a high spin Fe
3+-terpyridine (Fe-Tpy) complex as a T1 contrast agent. The anticancer drug demonstrated an accelerated release (~80% in 24 h) when exposed to an acidic medium, mimicking the tumor microenvironment but not under physiological conditions (pH 7.4). Additionally, these nanosystems increased drug retention > 65% in prostate cancer DU 145 cells after 10 h. Finally, a low IC
50 was determined to be 1 μg/mL, based on cytotoxicity assays conducted with the same cell lines, demonstrating the anticancer efficacy of this nanotheranostic system [
109]. Despite promising results, further studies should focus on the biodistribution of this novel nanoaggregate, which were not described in the study.
As an alternative, Mangadlao and co-workers developed gold nanoparticles, functionalized with PSMA-1 and loaded with a photosensitizer, Pc4, for fluorescent PDT [
108]. Gold nanoparticles presented a size of approximately 20 nm, a spherical design, and an absorbance peak at 680 nm, demonstrating that they can be used to selectively target a PSMA-expressing PC3pip cell model, in both in vitro and in vivo assays, with remission of the tumor 14 days after PDT [
108]. Overall, this strategy allows for an image-guided surgery of resectable tumors, as well as the elimination of nonresectable tumor ablation by PDT, without substantial side effects on the surrounding healthy tissue.
Considering particularly the unresectable tumors, a palladium radioisotope (103-Pd), used in clinical practice, was conjugated onto hollow gold nanoparticles (~150 nm) and assessed in vivo as brachytherapy seeds for a 5-week localized radiotherapy of prostate cancer [
110]. In this case, the nanoparticles were able to retain the tumor tissue for the entire treatment period, while no side effects nor accumulation were observed for preferential organs, such as liver and spleen [
110]. Since no functionalization was applied, this high retention rate was explained mainly by the large size of the particles and the intrinsic EPR effect of gold nanoparticles, which preferentially accumulate at tumor sites via passive targeting. Furthermore, and as seen in other works already described herein, the authors propose taking advantage of the gold nanocarriers as radiosensitizers to enhance the DNA damage by radiation [
110].
In another study, small 5 nm gold nanoparticles were conjugated with dithiolated diethylenetriamine pentaacetic acid (DTDTPA), to increase their stability and allow for a combination of CT imaging and radiotherapy [
111]. Briefly, these nanoparticles achieved moderate uptake by prostate cells, corresponding to passive accumulation in the tumor tissue, dependent on DTDTPA concentration, which protected the nanoparticles from opsonization and clearance by RES. In terms of therapeutic value, these nanosystems improved survival by 31%, compared to the group of mice receiving only radiation therapy [
111].
Despite the multiple advantages of those metallic nanosystems, numerous studies have also explored functionalization for the localized treatment of prostate cancer, in order to improve the bioavailability and target specificity of the nanoparticles. Recently, 40 nm gold nanocages were functionalized with neuropeptide Y (NPY), a ligand involved in the regulation of prostate cancer cell growth, using thiolated PEG [
112]. The cubic-shaped gold nanocarriers were assessed as intrinsic probes for imaging and tumor ablation via NIR light-based PTT, showing an absorbance between 802 and 806 nm [
112]. Additionally, the nanocages were able to stabilize the helical secondary structure and preserve the binding motif of the NPY, so that the receptor recognition ability was not compromised; in addition, the nanosystems were able to stimulate ERK activation, which indicates an interaction with the receptors from prostate cancer cells [
112]. Mainly, the authors have proposed that internalization occurred via two mechanisms: micropinocytosis (for aggregated particles above 300 nm) and clathrin-mediated endocytosis (for individualized nanoparticles). Lastly, in vitro testing showed that combining both the NPY-functionalized gold nanocages and NIR laser irradiation at 800 nm for 20 min decreased tumor cell viability, showing extensive necrosis [
112].
Another study focused on the design of a triple multimodal nanotheranostic, taking advantage of the intrinsic fluorescence of porphyrin-based nanosystems and combining both PTT and PDT modalities. This nanoporphyrin-based drug delivery system, which is also an inorganic PTT/PDT agent, could promote thermal ablation by NIR light-triggered activation and the formation of reactive oxygen species (ROS) (Lin2018). In addition, these nanoparticles were loaded with two Hsp90 inhibitors, 17-allylamino-17-demethoxygeldanamycin (17AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17DMAG), to decrease the levels of pro-survival and angiogenic signaling molecules induced by PTT, so that cancer cells would become more sensitive to this therapy [
113]. Similarly, the non-functionalized plain nanoparticles were able to accumulate in the tumor tissue while showing a residual presence in other organs, such as the intestine, liver, spleen, and lungs. In terms of in vivo studies, the 17AAG-loaded nanoporphyrin systems associated with light irradiation at low dose (0.5–1.25 W cm
−2 for 3 min) were more efficient compared to each individual treatment modality and did not cause any significant side effects [
113].
Considering the myriad of systems and strategies assessed for an early detection and treatment of prostate cancer, as well as for other tumors, there is a promising possibility of achieving synergistic effects from combining more than one therapeutic modality or imaging tools without potentiating toxic interactions.