2.1. Efflux Pump P-Glycoprotein and Drug Resistance

One of the most relevant resistance mechanisms of cancer cells is P-GP, a membrane protein which pumps out the drugs from the inside of cells to the extracellular space, reducing their therapeutic effect [18]. This protein, also known as ATP-binding cassette subfamily B member 1 (ABCB1) or ABC transporters, comprises a superfamily of efflux pump proteins with different members that may be involved in the MDR phenotype of tumor cells. In addition to P-GP, other members from this superfamily play an important role in the MRD phenotype in CRC and PAC, such as MDR-associated proteins (MRPs/ABCCs) and breast cancer resistance protein (BCRP/ABCG2) [9,19] and MDR1 [20,21]. P-GP is a 170 kD protein normally present in a variety of cells of the digestive system (e.g., common bile duct of the liver and pancreatic ducts), being highly expressed in the apical surface of epithelial cells [20]. However, the overexpression of P-GP in cancer cells is usually associated with a low therapeutic efficacy [22,23,24,25,26,27] especially in CRC. In this context, many nanoformulations have been designed to overcome the P-GP-mediated MDR phenotype in CRC and PAC (Table 1).

Mesoporous silica (mSiO2); pH responsive polyhistidine (PHIS); acriflavine (ACF); hypoxia-inducible-factor-1α (HIF-1α); d-α-tocopherol polyethylene glycol 1000 succinate (TPGS); doxorubicin (DOX); P-glycoprotein (P-GP); colorectal cancer (CRC); pancreatic cancer (PAC); photothermal therapy (PTT); poly lactic-co-glycolic acid (PLGA); nanoparticles (NPs); verapamil (VER); polyethylene glycol (PEG); paclitaxel (PTX); arginine-glycine-aspartic acid (RGD); bufalin (BU); hydroxypropyl-β-cyclodextrin (HP-β-CD); polyethylenimine (PEI); chlorin e6 (Ce6); ATP-binding cassette subfamily G member 2 (ABCG2); NF-κB decoy oligodeoxynucleotides (ODNs); photodynamic therapy (PDT); poly[N-(2-hydroxypropyl)methacrylamide]-poly(N,N-dimethylaminoethylmethacrylate) (pHPMA-b-pDMAEMA); antisense oligonucleotides (ASOs); camptothecin (CPT); epirubicin (EPR); alendronic acid and hyaluronic acid (AL–HA); irinotecan (IRI); nitric oxide-releasing DOX (NitDOX); polyethyleneimine/all-trans retinoic acid conjugates (PRA); poly(styrene-co-maleic acid) (SMA); gemcitabine (GEM); not specified (NS).

Despite the fact many P-GP inhibitors have been developed (e.g., verapamil, tariquidar, 11C-laniquidar, natural alkaloids or herbal natural products) [55,56,57,58,59], most of them showed significant limitations (systemic toxicity, insolubility, short blood half-life or rapid metabolization) [21,58,60,61]. The use of nanoformulations could overcome these drawbacks (Figure 1A). Recently, bufalin (BU), an antitumor drug that blocks P-GP-mediated resistance in CRC [62], was associated with vitamin E succinate-grafted-chitosan oligosaccharide with RGD peptide (arginine-glycine-aspartic acid) and TPGS. Compared to free BU, this combination showed improved antitumor activity (43% tumor volume reduction), pharmacokinetic profile and toxicity in resistant-CRC (LoVo/ADR cells)-bearing nude mice [31]. Moreover, the nanoformulation alone (i.e., without P-GP inhibitors) can overcome the P-GP-mediated MDR. For instance, treatment of PANC-1, a PAC cell line that overexpresses P-GP with DOX-loaded liposomes modified with phage fusion proteins for specific targeting, showed similar drug accumulation in presence and absence of verapamil [63,64]. Accordingly, this nanoformulation avoided P-GP-mediated expulsion and increased cytotoxicity of DOX (IC₅₀ 10-fold lower) [47]. Pan, et al. [65] overcame MDR mediated by P-GP in HCT8/ADR resistant cells using a large nanoformulation consisting of DOX-loaded poly(aspartic acid) NPs functionalized with TAT peptide and PEG, which is not a substrate of the efflux pump. In other cases, multifunctional NPs that combine several strategies at the same time to overcome the MDR phenotype have been used. For example, DOX-loaded gold nanorods coated with mesoporous silica (mSiO₂) used in CRC photothermal therapy (PTT) and chemotherapy have been improved by using pH-responsive polyhistidine (PHIS) and D-α-tocopherol PEG 1000 succinate (TPGS) to induce endocytic pathway escape and to inhibit P-GP, respectively [28]. These nanorods showed excellent results in male athymic nude mice bearing SW620/Ad300 cells, achieving a significant reduction in tumor volume (~3-fold) with low systemic toxicity. Interestingly, resistant CRC has also been treated with local PTT using gold nanorods coated with PEG and paclitaxel (PTX) nanocrystals coated with TPGS (all of them combined into a hydrogel). Promising results were obtained in the SW620/Ad300 cell line in vitro (~178-fold decrease in the IC50 of PTX) and in male athymic nude mice bearing SW620/Ad300 tumors [30]. The use of phosphatidylserine (PS) lipid nanovesicles with encapsulated PTX showed a synergistic effect against the chemoresistant HCT15 cell line overexpressing the MDR1 gene product P-GP, both in vivo an in vitro. These NPs induced cell cycle arrest at G2/M phase, downregulated ki-67, Bcl-2, and CD34, upregulated caspase 3, reduced the systemic toxic effects of PTX and no inflammatory response was reported [66].

Figure 1. Representative scheme of nanomedicine strategies against drug resistance in pancreatic cancer. (A) Interactions of nanoformulations with MDR mechanism in pancreatic cancer (PAC) cells. Drugs carried by NPs may overcome MDR mechanisms of PAC cells mediated by efflux pumps via the inhibition of drug efflux, decrease in the expression of the transporter proteins, and increased accumulation of drug inside the tumor cells. (B) Exploiting the overexpression of EGFR to overcome resistance mediated by RAS mutations in PAC cells. PAC cells overexpress EGFR, which can be targeted by NPs conjugated with mAbs (Cetuximab/Panitumumab) (see below). This enables increased internalization of the chemotherapy drugs, thus leading to cellular apoptosis, even in RAS-mutated cancer cells.

2.2. Other Efflux Pumps

Breast cancer resistance protein (BCRP/ABCG2), also known as mitoxantrone resistance protein (MXR) or placenta ABC protein (ABC-P), is a small member of the ABC superfamily (~75 KDa) with a more limited number of substrates (e.g., amptothecin [CPT], tyrosine kinase inhibitors and methotrexate) and inhibitors (e.g, imatinib or poloxamines) compared to P-GP [67]. Although BRCP/ABCG2 is usually highly expressed in hematological cancers, it has also been found to be overexpressed in CRC and PAC [68,69]. In fact, 72% of pancreatic cell lines from the Cancer Cell Line Encyclopedia (CCLE) overexpressed this protein [69]. Nanoformulations such as irinotecan-loaded liposomes with benzoporphyrin derivative for photodynamic therapy (PDT) were able to overcome ABCG2-mediated resistance, decreasing tumor volume from 70% to 25% in mice bearing pancreatic tumors [41]. PDT has also been used with polymeric NPs with PEG and polyethylenimine (PEI) in pancreatic cells with variable expression levels of ABCG2 (+AsPC-1 and -MIA PaCa-2 and induced +MIA PaCa-2), increasing the intracellular concentrations of the photosensitizer through reduction of its efflux by ABCG2 [37]. On the other hand, pegylated poly-lactic-co-glycolic acid (PLGA) NPs with verapamil and SN38 (active form of CPT) showed no significant differences in drug cytotoxicity in HT29 CRC cells, but a significant decrease in ABCG2 mRNA expression (2.277-fold) in comparison with the free drug (4.793-fold) was observed [35]. In addition, microbubbles containing porphyrin/CPT-floxuridine for chemotherapy, PDT and imaging, generated NPs suitable for tumor therapy after exposure to ultrasound. This treatment significantly reduced the expression of ABCG2 in HT29 cells by increasing the concentration of CPT, inducing an in vivo growth inhibition (90%) of the tumor without recurrence [70].

On the other hand, the MRP1/ABCC1 and MRP3/ABCC3 proteins, which have a similar molecular weight (~190 KDa) and structure, contain a different n-terminal region in relation to P-GP [71,72,73]. Drug substrates of MRP1 and P-GP are similar except for taxanes, which are only P-GP substrates [73]. The spectrum of molecules transported by MRP3 is limited [74] In fact, MRP1 is the protein mostly involved in the therapeutic failure resulting from MDR [75]. Some nanoformulations have been designed to specifically overcome efflux pumps, including MRP. Nitric oxide-releasing DOX (NitDOX) was loaded in liposomes to treat HT29 cells resistant to DOX mediated by MRP1 and P-GP. Nitration of both proteins significantly reduced their activity [48]. In addition, pegylated liposomes loaded with epirubicin and antisense oligonucleotides (ASOs) against MDR1, MRP1 and MRP2 increased the antitumor activity of the drug in mice bearing CRC (CT26 cells), while administration of ASOs alone did not show significant differences between resistant and non-resistant tumors [51].