DOX is commonly used to treat various types of cancer, including BC. However, despite wide use, it has well known undesirable side effects, such as cardiotoxicity (55). Various types of CPP have been coupled to a variety of NSs, including liposomes, gold, dendrigraft, iron oxides and polymeric structures. These NSs carrying DOX and/or other anticancer drugs have been tested in vivo and in vitro to improve delivery and reduce side effects. Below, the present review briefly describes some of the NSs that have been coupled to CPPs.

Hydrophobic CPP PVF coupled to a 100-nm liposome was evaluated in MCF-7, MDA-MB-435S, MCF-7/Adr (Adriamycin-resistant human mammary adenocarcinoma) and 4T1 murine BC carcinoma cell lines. Even when the cell survival rate was higher in cells treated with the NS in vivo, the NS decreased the tumor weight up to 0.4 g compared with the control (0.9 g) and free DOX (0.7 g), while keeping the mice at a healthier weight compared with the free DOX-treated mice (41).

With the ability to permeate the blood-brain barrier, the HIV-derived TAT CPP was evaluated in MDA-MB-231 cell lines and metastatic in vivo tumors using gold NPs coupled with PEG as the carrying vehicle for DOX. In vivo cell uptake increased 4.8-fold compared with that of the control, while DOX half maximal inhibitory concentration (IC₅₀) decreased 80% compared with that of free DOX. Furthermore, no significant adverse effects were found when the DOX dose of the NS ranged from 1 to 5 µg. However, at 10 and 15 µg DOX, the mice exhibited significant weight loss, which a common side effect of this drug. Also, the development of ascites and peripheral edema was found in the mice, which a relatively common adverse effect of DOX metabolism. These results suggest further targeting with this NS should be achieved before moving into clinical trials (43).

A gold nanostar coupled with mesoporous silica (MS), TAT fragment and the photosensitizer drug protoporphyrin IX was used as a theranostic system. Also, Raman detection for diagnostics, photodynamic therapy (PDT) and cytotoxicity were evaluated in the BT-549 cell line. Through Raman imaging, it was determined that TAT CPP activity allowed cell penetration while maintaining cell viability, although once PDT was applied, the cell viability was decreased (56). A similar study used an NS containing TAT and gold NPs along with anti-HER2 antibody for increased specificity and surface-enhanced Raman spectroscopy, which was assessed for DOX release rate into SK-BR-3 cells. After 24 h, in vitro cell viability was decreased by 39.48% compared with that of the control (57). Another study designed a synthetic peptide containing TAT, L-lysine residues, fusogenic GALA peptide and cell targeting peptide (DMPGTVLP) with stearic acid to improve condensation and stability to a dioleoylphosphatidylethanolamine (DOPE)-based nanoliposome. This NS was evaluated against MCF-7 cells delivering an siRNA targeting BCL2 mRNA. The chimeric peptide used in this NS increased cell uptake, and BCL2 silencing was decreased by ~80%. However, cell viability was decreased by only 20% (58).

The size of the NS directly impacts tumor accumulation and diffusion abilities. Keeping this in mind, researchers developed an NS that could be decreased in size by the action of metalloproteinase-2, while keeping high specificity for triple-negative BC (TNBC). Gelatin and dendrigraft polylysine were used as a carrying vehicle and stabilization agent, while DOX was used as the anticancer agent in 4T1 cells. As expected, in vitro cell uptake was increased from 15 to 40%, which translated into a higher inhibition rate. In vivo, there was an ~3-fold decrease in tumor volume and a 4-fold decrease in tumor weight compared with that for free DOX treatment. Furthermore, no difference in mouse bodyweight was reported, suggesting the relative safety of this NS (59).

A nanoliposome-based system carrying the arginine-rich amphiphile CPP and DOX or paclitaxel (PTX) was tested in MCF-7 cells. After 3 h, cell uptake had increased 5-fold with rhodamine B (a hydrophilic dye) and 30% with Nile red (a hydrophobic dye) to show this dual amphiphile activity. The drug uptake and effectiveness of the liposome improved with the NS compared with the use of free drugs. In vitro, cancer cell viability was decreased by 50 and 25% in the CPP-DOX-NS and NS-PTX groups, respectively (60). These results show a significant difference between NSs with CPPs and those that do not have them coupled.

The QLPVM CPP was attached to a 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE)-PEG-based nanoliposome carrying tamoxifen (TAM) and DOX, evaluated in MCF-7 cell tumor-bearing mouse models. In vitro cell viability was dose-dependent for the NS. However, cell viability was lower than that in the NS-free TAM and NS-free DOX groups. In vivo tumor inhibition with the combined NS showed the best results. In a synergistic effect, tumor volume decreased 7-fold compared with that of the control, with similar results in tumor weight and no noticeable bodyweight loss or cardiac toxicity (61).

A disadvantage of drug therapy is the existence of drug-resistant tumors. Thus, the chimeric R8 CPP was fused with an apoptotic peptide to create a co-delivery nanocomplex in MCF7-DOX resistant cells. The nanocomplex consisted of binding several wild-type p53 proteins, chimeric CPPs and p53 DNA. Assembly was enabled by preparing a mixture of these components and incubating them at 37°C for 30 min to allow the formation of the nanocomplex. There was a 36-fold in vitro increase in apoptosis in the synergistic NS compared with that in free DOX, leading to a 97.9% decrease in cell viability. In vivo, there was a significant tumor volume and weight decrease when the mice were treated with the NS. Tumor volume decreased ~10-fold, while tumor weight was 9-fold lower at 21 days post-injection (62).

The R₇ CPP was coupled in a polylactic-co-glycolic acid-PEG polymer with folic acid and the cell-cycle anticancer agent, vincristine sulfate (VCR). Cell uptake mediated by R₇ was not significantly different from cell uptake mediated by folic acid and its receptors. However, cell viability was decreased to 30% when 5 nM VCR was added, compared with 60% cell viability without VCR. These results were further confirmed by determining an increase in MCF-7 cell percentage in the G2/M arrest phase from 12.28±1.74% to >33% (63).

A DSPE-PEG-based nanoliposome coupled with R₉ was developed to deliver cabazitaxel in the 4T1 cell line and murine models. Cell uptake was increased 1.78-fold after 8 h of exposure compared with the control. In vitro cytotoxicity was enhanced 223-fold to 0.03 µg/ml due to NS activity. In vivo, the NS localization in the lymph nodes was reported to increase 1.73-fold at 24 h post-injection. This finding demonstrates a penetrating activity with long retention times. Also, primary tumor growth decreased 75.3%, while tumor inhibition in the lymph nodes was reported at 89.1%, showing that this NS can inhibit metastasis. The size of this NS was 13 nm; NSs with sizes <30 nm have been reported to have better access to lymph nodes (64–66).

Vesicles extracted from red blood cell membranes and coupled to iRGD peptide were used as biological NSs carrying PTX. In vitro cell uptake in 4T1 cells was higher in the NS compared with that in the control. However, in vitro cell viability was similar compared with that of free PTX. In vivo, the NS decreased tumor growth to 8.5%, 5.6-fold less compared to the control (67). In a similar study, the iRGD CPP coupled to iron oxide nanoworms was used as an NS to inhibit or lower BC metastasis to the brain, targeting MDA-MB-231 and 4T1 ×enografts in 231Br and 4T1-BR5 mouse models. Brain imaging showed that 30 days post-injection, the number of tumors was decreased by 60%, and their size had decreased 5-fold compared with the controls. However, researchers found a tight window of 12 days for this metastasis decrease (68).

The pentapeptide CALNN CPP coupled with gold NPs was used to deliver linalool (a monoterpenoid derived from plants) and evaluate its anticancer activity in MCF-7 cells. In vitro cytotoxicity was reported at >80% after 48 h of treatment, while clonogenicity was inhibited. In vivo, the NS conferred no statistical change in mouse health, suggesting the safety of the NS for further study. Unfortunately, the study did not include in vivo anticancer activity assays (69).

CREKA, a linear pentapeptide designed to bind to fibrous protein in the tumor microenvironment, was fused to the CF peptide (a CPP for nucleus translocation) using a polylactic acid-based NS as the vehicle. This NS was designed for targeted delivery of the anticancer drug erlotinib into MDA-MB-231 cell lines to combat drug resistance in TNBC. Cell uptake was increased 2.3-fold when the pH of the assay was similar to that of the tumor microenvironment. In vitro cell viability decreased <20% in the pH simulating the tumor microenvironment, compared with that in the physiological pH, which maintained 50% viability. In vivo, a 2.8-fold tumor decrease was observed in mice treated with the NS, while their body weight was maintained, suggesting an effective method for TNBC tumor treatment that lacked adverse effects (70).

The tLyP-1 CPP, which specifically targets tumors, was used to develop an NS targeting MDA-MD-231 cells. The polymeric NPs consisted of HA and D-α tocopheryl succinate as a vehicle. The NS carried the anticancer drug docetaxel. Cell uptake compared with that of the control increased 2.36-fold, while in vitro, cell cytotoxicity was higher in the NS with tLyP-1. In vivo tumor weight decreased 3.5-fold compared to the control (71).

The PEGA-pVEC CPP that targets BC cells and tumors was coupled to an NS of colloidal MS as a vehicle for the anticancer agent epigallocatechin-3-gallate (EGCG) in MCF-7 cells. In vitro cell inhibition reached 100% at a 100 µg/ml concentration. In vivo tumor inhibition of up to 89.66% was reported compared with 69.9% in the control group in murine models (72). In a similar study, PEGA-pVEC was used to develop an MS and HA NS as a carrying vehicle for DOX and an siRNA directed against the BC overexpressed connective tissue growth factor gene. The NS was tested in drug-resistant MDA-MB-231 cells. In vitro inhibition increased from 59 to 80% compared with the control. In vivo, the tumor inhibition increased up to 60% compared with the control (73). A similar study developed a self-aggregating nano-gel based on protamine aggregates carrying EGCG as an anticancer agent to MDA-MB-231 cells and xenografts. In vitro, there was a 15-fold increase in cytotoxicity compared with the control, while in vivo tumor inhibition was 80% compared with the control (74).

The chimeric arginine-glycine-aspartate CPP was evaluated in MDA-MB-231 cells using gambogic acid (GA) as an anticancer agent and nanostructured lipid nanocarriers. When cells were treated with a 2 µg/ml concentration, in vitro cell viability was decreased to <20% compared with that of the control. However, the best inhibition rate and tumor size decrease were achieved with the NS carrying RGERPPR alone, suggesting that only one CPP can benefit a single NS (75).

An NS, delivering a novel, non-disclosed siRNA consisting of superparamagnetic iron oxide NPs (SPIONs) functionalized with PEG, and cationic polymers, such as chitosan and L-arginine with CPP gH625, was designed and evaluated against the MDA-MB-231 cancer cell line. After 4 h of exposure, the NS penetrated the cells aided by the gH625 peptide, and the MDA-MB-231 cancer cell line genes were downregulated due to the interference mechanism of the siRNA. At 72 h, there was cell growth inhibition of nearly 80% (51). gH625 in an NS SPION couple was also tested in MDA-MB-231 cells as a theranostic system. Cell uptake increased 3-fold in the NS compared with that in the control assay (76).

The Twist gene is a transcription factor for an epithelial-mesenchymal transition that is heavily involved in the metastatic activity of tumors. Therefore, a nano-corona NS consisting of a DOPE NP carrying an siRNA against the Twist gene, the R₉ for tumor penetration, and coated with human serum albumin for immune system camouflage, was designed and evaluated in 4T1 cells. The nano-coronas were labeled with IR-780 dye for combination therapy: Twist gene silencing and tumor growth inhibition using photothermal therapy for thermal ablation. Cell uptake indicated a higher NS translocation in the cytoplasm after 24 h of incubation; the cell migration rate decreased up to 66%. The cell viability index was decreased by 90% when treated by photothermal therapy. In vivo experiments showed the ability of the NS to penetrate tumors; tumor inhibition progression was 83.6% and metastasis inhibition was 92.2% after 13 days of combination therapy (77).

This CPP was used to develop a lipid-based nanobubble system carrying an siRNA against an epidermal growth factor. The main objective was to evaluate the NS when applying ultrasound irradiation inside tumor cells to enhance the siRNA anticancer activity targeting MDA-MB-231 cells. Cell inhibition was increased from 48 to 72 h, while in vivo tumor growth was decreased by 42.08% (78).

The fusion of a HER2 antibody mimetic-affibody and CPP Tat-Mu was used to deliver an anti-tissue factor using an shRNA. The NS was a cationic N,N-di-n-hexadecyl-N,N-dihydroxy ethyl ammonium chloride-based nanoliposome used as a vehicle to MDA-MB-231 cells and xenografts. In vitro, cell uptake increased 7-fold compared with that of the control, while there was a significant tumor size decrease, to <10% of the original size, in vivo (79).

The TT1 CPP has specificity against p32, a mitochondrial essential tumor regulator protein. Iron oxide NPs were the vehicle for the TT1 CPP fused with the proapoptotic peptide [D(KLAKLAK)2] (inducing apoptosis through the activation of caspase-3) and tested in MCF10CA1a and 4T1 cell lines. In vitro cell uptake increased 4-fold, while a tumor size decrease of 50% was observed in vivo (80). Another study consisted of a cholesterol-based nano-micelle carrying TT1 plus two anticancer components, siRNA targeting PDL-1 and indoleamine 2,3-dioxygenase inhibitor (1-methyl tryptophan), that led to the activation of cytotoxic T lymphocytes against the 4T1 cell line. After 4 h, cell uptake was 16.3-fold higher than that of the control assay (81).

Ephrin type-A receptor 2 (EphA2) is a transmembrane protein whose overexpression has been linked to carcinogenesis, metastasis and a poor clinical prognosis. The YSA peptide, an ephrin mimetic bound with EphA2, was fused with a penetratin-derived CPP. DSPE-PEG nanobubbles were used as the vehicle for an anti-Myc siRNA acting as an anticancer agent. The in vitro cell uptake was higher with this NS treatment. In vivo tumor growth inhibition after 24 days of treatment was decreased to 31.2% compared to the control, while no significant body weight loss was found in the mice. The NPs with YSA peptides improve therapeutic effects in vivo (82).

A urokinase activatable CPP was developed to ensure tumor specificity for in vivo targeting of 4T1 cell tumors in murine models. In addition, silver NPs were coated with PEGylated neutravidin as a vehicle and for immune system stealthiness. However, in vivo distribution was determined only in tumor tissue, without fluorescence in healthy tissue (83).

The difference in pH between the tumor microenvironment and the healthy extracellular matrix has been exploited along with CPPs to improve the specificity of the NS. Nanopolymer micelles masked with PEG-lactic acid were used as a vehicle for the RACPP, containing a pH-sensitive sequence, and PTX as an anticancer drug targeting 4T1 cells and 4T1-BALB/c tumor xenografts models. Cell uptake was increased 3-fold, while in vitro IC50 was decreased from 1.595 to 1.035 µg/ml. In vivo, tumor size decreased by ~50% at 16 days post-injection, while the mouse survival rate increased 1.5-fold (20).

In another case, the pH-sensitive MAP CPP was used with the highly pH-sensitive histidine-glutamate (HE) oligopeptide (HEHEHEHEHEHEHEHEHEHE) coupled to glutathione-S-transferase acting as a cargo protein. The delivery activity was measured in MDA-MB-231 cells and xenografts through fluorescence imaging. At an acidic pH simulating the tumor microenvironment, there was a 3-fold increase of fluorescence in vitro. In vivo results showed that at 6 h post-injection in mice, the system was localized mainly around the xenograft tumor. These results further show the advantage of using the pH of the tumor microenvironment for intelligent drug delivery systems (84).

Using a different approach, a thermosensitive NS was developed using a heat-activatable CPP after mild thermal stimulus at temperatures ranging from 37 to 42°C, which facilitates drug delivery. The NS consisted of a nanoliposome that immediately encapsulated drugs in heating tissue or organs. These were coupled with a penetratin-derived CPP, the NGR peptide and DOX as the anticancer agent against MCF-7. In vitro cellular uptake was increased 5-fold compared with the control, while in vitro cytotoxicity of the preheated NS showed an increase of 1.5-fold compared with the NS without preheating treatment. However, both cases showed increased cytotoxicity compared with that of free DOX. Similarly, in vivo tumor inhibition was increased in the preheated NS. Also, there was an ~9-fold tumor volume decrease with the preheated NS compared with the control, with no apparent body weight decrease (42).