Introduction

According to World Health Organization, 8.8 mln people died from cancer in 2015 and it was one of the leading causes of death worldwide [1]. Although during the last two decades mortality rates decline, they still remain high [2]. Therefore, it is very important to improve cancer diagnostics and enhance the efficacy of anticancer therapy. To ensure the therapeutic efficacy of an anticancer drug, sufficient concentration of the compound must be achieved in tumor cells. It becomes a challenge for the drug developers due to certain characteristics of the tumor microenvironment [3]. An increasing attention is given to the transport of drugs to tumors.

Due to increased extracellular acidity of tumor, basic drugs tend to ionize. Positive charge limits drug ability to permeate cellular membrane and reach the target site [4]. The efficacy of chemotherapy is also associated with MDR which is partially caused by various drug efflux proteins, that transport drug molecules from the cytoplasm to the extracellular fluid [5, 6]. Multidrug resistance protein-1 (MRP-1/ABCC1) has been shown to be associated with MDR in stage I and II hormone positive breast cancer (n = 516). Patients who administered cyclophosphamide, methotrexate, and fluorouracil and who had increased expression of MRP-1 experienced increase in relapse rate and mortality when compared to those patients, who had negative MRP-1 expression in tumors [7]. In other clinical study (n = 59), high phosphoglycoprotein (P-gp/ABCB1) expression level was associated with a poor prognosis of a disease and decreased length of progression-free survival [8]. It is important that cell sensitivity to chemotherapy and the mechanisms of resistance vary between different types of cancer and different cell lines of the same type of cancer. Kibria et al. investigated the sensitivity of 15 different cell lines to doxorubicin. They found that the sensitivity of cancer cells to doxorubicin was not always proportional to the intracellular concentration of doxorubicin. Also, the inhibition of P-gp in certain cell lines significantly increased cytotoxicity of doxorubicin, while in other cell lines, no effect was observed. This means that, besides P-gp, there are some other mechanisms that determine drug resistance [9]. Various types of tumors differ in the rates and mechanisms of chemoresistance. For example, a characteristic feature of pancreatic cancer is a very rich stroma with a high number of fibroblasts and macrophages. It was found that these cells contribute to chemoresistance by secreting various growth factors which leads to increased cell survival and proliferation [10, 11]. Furthermore, cancer stroma is like a physical barrier to drug penetration. High number of stroma cells and increased interstitial pressure cause the constriction of blood vessels and limited drug penetration into target site [12]. Also, pancreatic cancer is usually highly hypoxic. Hypoxia may increase chemoresistance by activation of certain signaling pathways [13]. Pancreatic cancer is very poorly vascularized [14]. It is considered to be one of the most hypoxic types of cancer [14]. Also, increased expression of drug efflux transporters multidrug resistance protein-5 (MRP-5/ABCC5) [15], multidrug resistance protein-5 (MRP-8/ABCC11) [16], and human equilibrative nucleoside transporter-1 [17] contributes to chemoresistance to gemcitabine and 5-fluorouracil.

In order to overcome these problems of inefficient cancer chemotherapy, pH modulators (vacuolar-H+-ATPase (V-ATPase) inhibitors, carbonic anhydrase (CA) inhibitors, sodium-hydrogen exchanger-1 (NHE-1) inhibitors), inhibitors of MDR proteins P-gp, MRP-1, and breast cancer resistance protein (BCRP/ABCG2)), nanocarrier systems, and physical methods (sonoporation, electroporation) are being developed and widely investigated.

Tumor microenvironment

There are some significant differences between normal and tumor tissues and one of the main discrepancies is a pH gradient. Because of anaerobic metabolism in tumor tissues, extracellular fluid is more acidic than in normal tissues [18]. It was estimated that extracellular pH in tumor varies between is about 6.8 [19, 20]. Activation of oncogenes, hypoxia-inducible factor-1 activation arises in cancer cells, and this leads to induced expression of glycolytic enzymes [21]. Upregulation of glucose transporters occurs, as well [22]. Therefore, cellular energy metabolism shifts from oxidative phosphorylation towards anaerobic glycolysis even in the presence of oxygen. This phenomenon is called the Warburg effect and was discovered by Otto Heinrich Warburg in 1920s [23]. High rate of glycolysis results in increased lactic acid production and various transporters, such as V-ATPase, NHE, monocarboxylate transporter, extrude protons into the extracellular tissue, thus increasing its acidity [24, 25].

It is known that neutral molecules penetrate cell membrane easier than positively or negatively charged ions [26]. According to pH-partition theory, at lower pH basic drugs, e.g., doxorubicin, undergo ionization, therefore, the penetration of these compounds declines and their therapeutic efficacy decreases (Fig. 1) [18, 27]. This is called “ion trapping” phenomenon. The same problem is typical with other basic compounds such as anthracyclines, anthraquinones, and Vinca alkaloids. Weakly basic drugs also tend to accumulate in lysosomes and endosomes that have an acidic lumen [28, 29].

Fig. 1
figure 1

Mechanism of acidity-related chemoresistance. Due to increased acidity in cancer cells protonation of basic drugs occurs thus limiting their ability to penetrate through the cell membrane

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There are two main strategies to reduce this acidity-related chemoresistance. One of them is increasing pH of extracellular milieu by basic substances, such as sodium bicarbonate [27, 30]. Robey et al. investigated that oral administration of 200 mM sodium bicarbonate slightly increased extracellular pH from 7.0 to 7.4, whereas did not affect intracellular pH in murine breast cancer models [31]. Other study showed that oral administration of sodium bicarbonate in mice may enhance the activity of basic anticancer drugs, such as doxorubicin [27]. However, it is associated with a risk of metabolic alkalosis, hypernatremia, electrolyte imbalance, and other side effects [32]. Therefore, other more modern approaches, based on the inhibition of proton transporters, CAs or ATP-binding cassette (ABC) transporters are being developed. These enzymes and transporters contribute to chemoresistance by pumping protons from cytoplasm to extracellular tissue and acidic vesicles, thus increasing extracellular acidity, and by mediating drug efflux out of cancer cell (Fig. 2). Compounds that inhibit proton pumps prevent proton transport from the cell cytoplasm to extracellular milieu and suppress the acidification of the extracellular tissue [33].

Fig. 2
figure 2

Transporters and enzymes involved in multidrug resistance. Proton transporters V-ATPase and NHE extrude protons out of cells thus decreasing pH of extracellular milieu; CA IX contributes to the extracellular acidity by carbon dioxide hydration resulting in bicarbonate ion and proton release. ATP-binding cassette transporters P-gp, MRP-1, and BCRP mediate anticancer drugs efflux out of the cell. Abbreviations: V-ATPase, vacuolar-H + -ATPase; NHE, Na+/H+ exchanger; CA IX, carbonic anhydrase IX; P-gp, phosphoglycoprotein; MRP-1, multidrug resistance protein-1; BCRP, breast cancer resistance protein

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pH modulators

One of the strategies to increase the penetration of basic drugs into the cells is to reduce the extracellular acidity in tumor tissue. For this purpose, various pH modulators, such as proton pump inhibitors (PPIs), CA inhibitors, or NHE inhibitors are being applied.

V-ATPase inhibitors

V-ATPase is a proton pump which regulates H+ transport across cell membrane and maintains low pH within endosomes and lysosomes [34]. It is located in the membrane of lysosomal vesicles and in the plasma membrane of certain cells. This enzyme pumps H+ from the cell into the interstitial fluid, thus maintaining acidic extracellular pH and transport H+ from cytosol to cellular vesicles [35].

It is well known that in some tumors the activity of several isoforms of V-ATPase is increased [36,37,38]. High activity of this transport protein is associated with a poor prognosis of the disease [39]. Compounds that inhibit V-ATPase lead to pH increase in extracellular milieu and acidic vesicles [40]. Thus, basic drugs can easier penetrate into cancer cells [41]. V-ATPase inhibitors also reduce basic drug sequestration and neutralization within lysosomes and their extrusion out of cells via exocytosis [42]. It leads to decrease of drug trapping, enhanced delivery to their target site, and improved cell sensitivity to chemotherapy. Lee et al. found that siRNA-induced inhibition of V-ATPase leads to the reduced intracellular pH and increased cytotoxicity of paclitaxel in chemoresistant epithelial ovarian cancer cells [39].

Plecomacrolide antibiotics bafilomycins and conacanamycins are the earliest known V-ATPase inhibitors. It was shown that bafilomycin A1 effectively inhibits V-ATPase, thus increases extracellular pH in cancer cell cultures [43]. Similar results were found with concanamycin A. Kiyoshima et al. estimated that this compound inhibits the acidification of cellular vesicles and reduces proliferation of oral squamous carcinoma cells [44]. Nevertheless, none of these compounds was introduced to clinical trials.

Another well-known group of V-ATPase inhibitors is PPIs. Recent studies show that drugs, such as omeprazole, lansoprazole, and pantoprazole, reverse MDR by reducing extracellular acidity [39, 41, 45, 46]. PPIs tend to accumulate in acidic cell compartments where they are activated by the protonation of basic nitrogen atoms [47]. In this way, they might specifically be active in acidic tumor tissues. Patel et al. found that pantoprazole increased endosomal pH and nuclear uptake of doxorubicin within mouse mammary sarcoma cells and tumor tissue [46]. There is evidence that omeprazole pretreatment in combination with paclitaxel reduces tumor growth in chemoresistant epithelial ovarian cancer mice models compared to paclitaxel alone [39]. Similar results were reported by Luciani et al. [48].

It is important to emphasize that simultaneous treatment with PPIs and antitumor drugs did not potentiate the efficacy of chemotherapy. Authors explain this phenomenon by the competition of drugs against each other for cellular uptake [48]. However, the data about the disruption of intracellular and extracellular pH gradient caused by PPIs is controversial. Linder et al. detected no difference in intracellular and extracellular pH after 24 and 48 h of cancer cells treatment with PPI. Furthermore, increase in intracellular pH and decrease in extracellular pH were determined in these cells after 72 h of treatment with PPI [49]. These data suggest that there may be some other mechanisms through which PPIs enhance the cytotoxicity of anticancer drugs. Also, there is evidence that co-administration of PPIs with certain anticancer drugs may reduce some side effects of chemotherapy. Recently, Ikemura et al. investigated that PPIs can ameliorate the nephrotoxicity of cisplatin by inhibiting organic cation transporter 2 [50]. Furthermore, there is evidence that PPIs not only improve the efficacy of chemotherapeutics, but also exert anticancer activity themselves. Among these drugs, lansoprazole has shown the most potent cytotoxicity [51].

Because of high potential for the application in chemotherapy, PPIs gained a great interest among researchers. On the basis of existing structures, novel bisbenzimidazole derivatives were developed. These compounds showed to be potent V-ATPase inhibitors and demonstrated high antiproliferative activity against breast cancer cells [52] and are among the most promising transport modulators of basic drugs.

CA inhibitors

CA is a transmembrane zinc metalloprotein which is involved in pH homeostasis of various tissues. This enzyme catalyzes the reversible hydration of carbon dioxide to bicarbonate [53]. 16 α-CA isoforms exist in mammals. Two of them—CA IX and XII—are associated with cancer development and progression [54, 55]. According to Robertson et al., inhibition of CA IX expression results in a delay of cancer cell growth and reduction of cell survival under normoxia and hypoxia [54]. Although both isoforms are found in normal tissues, such as the gastric mucosa, duodenum, or kidney, their gene expression is highly increased in many types of tumors [56, 57]. Therefore, they are attractive targets for anticancer therapy.

It is hypothesized that inhibition of CA IX and XII reduces extracellular acidification, therefore enhancing basic drug delivery into tumor tissue. There are some preclinical studies confirming this theory. Gieling et al. found that inhibition of CA IX by acetazolamide enhances uptake and toxicity of weakly basic doxorubicin, but reduces weakly acidic melphalan penetration into cells [58].

Several CA IX and CA XII inhibitors have been developed and tested as transport modulators. However, most of them are still in preclinical studies and the results are controversial. SLC-0111, also known as U104, is a benzenesulfonamide derivative and highly selective inhibitor of CA IX and CA XII. It was shown that U104 significantly increased the efficacy of paclitaxel in orthotopic breast cancer mice models [59]. In contrast, Riemann et al. assessed that although U104 reduced cancer cell proliferation and increased apoptosis, it did not improve the cytotoxicity of daunorubicin and cisplatin in prostate cancer cells [60]. Phase I clinical study was designed in order to evaluate the pharmacokinetic profile, safety, and efficacy of U104 in anticancer therapy (clinicaltrials.gov, NCT02215850). However, the results have not been published yet.

For higher therapeutic efficacy, CA inhibitors can be incorporated into nanocarrier systems. Janoniene et al. loaded porous silicon nanoformulations with doxorubicin and conjugated with selective CA IX inhibitor VD11-4-2 [61]. It enhanced target drug delivery towards tumor tissue and improved doxorubicin penetration into cancer cells. VD11-4-2 also enhanced drug loading efficacy into nanoparticles and improved drug release profile by reducing the premature release of doxorubicin. Besides an increase in intracellular drug concentration in tumors, these systems also reduce the effect on other tissues thus reducing toxicity. Therefore, this field is currently very widely investigated and show promising results.

NHE inhibitors

NHE is a ubiquitous proton transporter that mediates Na+/H+ exchange across the cell membrane. It extrudes protons out of the cell and transports Na+ into the cytoplasm. As cellular pH regulators, NHEs also contribute to MDR. Inhibition of these transporter proteins increases basic drug penetration into tumor tissue. Thirteen isoforms of NHE exist in humans. NHE-1 is of particular interest in oncology because it is involved in cancer cell migration [62] and metastasis [63]. NHE-1 is found in many normal tissues and also is upregulated in various tumors, such as gastric [64] and breast cancer [63], hepatocellular carcinoma [65] or glioblastoma [66]. Previous studies showed that knockdown of NHE-1 results in increased sensitivity to doxorubicin in T cell acute lymphoblastic leukemia cells [67].

The first known NHE inhibitor was potassium-sparing diuretic amiloride, discovered in 1965 [68]. A few decades later, more potent amiloride derivatives specific to NHE-1 were synthesized. 5-(N-ethyl-N-isopropyl)amiloride (EIPA) is amiloride analog which is 200-fold more effective in inhibition of NHE. Some studies show that EIPA may reverse doxorubicin resistance in cancer cells. Pannocchia et al. found that EIPA significantly increased doxorubicin accumulation in doxorubicin-resistant colon cancer cells while the addition of monensin (NHE activator) significantly reduced intracellular doxorubicin concentration [69]. Miraglia et al. showed that inhibition of NHE by EIPA leads to the reduction of intracellular pH and thus increases doxorubicin concentration in colon cancer cells [70]. On the contrary, activation of NHE by phorbol 12-myristate increases intracellular pH and decreases penetration of doxorubicin into cells.

Recently, more powerful and highly selective NHE-1 inhibitors cariporide, zoniporide, and eniporide were developed. Although they showed good tolerability in humans, all the clinical trials performed were oriented in the field of cardiology because of their cardioprotective effects [71, 72].

ABC transporters

Another reason for MDR is the activity of ABC transporters. They are transmembrane proteins involved in self-defense mechanisms. These proteins actively pump various endogenous molecules and xenobiotics out of the cell. The main transporters that are linked to the resistance of many structurally unrelated anticancer agents are P-gp, MRP-1, and BCRP [73]. The substrates of these transporters include numerous anticancer agents from various groups (Table 1).

Table 1 Substrates of ABC transporters
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ABC transporters are normally found in many various organs such as the kidney, liver, testes, intestine, and physiological barriers [95]. Usually, their expression in tumor tissues is highly increased [96, 97]. It was shown that inhibition of these transporters may enhance delivery and efficacy of anticancer drugs [78].

P-gp inhibitors

The most widely studied ABC transporter is P-gp. It is a 170-kD transmembrane protein. P-gp overexpression causes chemoresistance against many anticancer agents, such as paclitaxel, doxorubicin [98], daunorubicin [99], etoposide [100], or vinblastine [101].

There are three generations of compounds that inhibit P-gp (Table 2). First generation includes verapamil, quinine, and cyclosporine A. These are pharmacologically active compounds, approved for various cancer unrelated indications. In 1989, it was found that verapamil competitively inhibits P-gp and enhances doxorubicin, colchicine, and vincristine retention within leukemia cells [102]. Cyclosporine A was found to enhance distribution, retention and cytotoxicity of doxorubicin and mitoxantrone in cells overexpressing P-gp, MRP-1 and BCRP [103]. However, during some studies, it was noticed that these drugs lack efficacy [104] or may improve the toxicity of chemotherapy and cause various adverse events [105, 106].

Table 2 Generations of P-gp inhibitors
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In order to reduce toxicity, first-generation P-gp inhibitors were modified using chiral switching, and second generation P-gp inhibitors were developed. Drug binding to P-gp is not stereospecific; thus, isomers of P-gp inhibitors maintain their inhibitory effect and ability to reduce MDR. Dexverapamil, the R-isomer of verapamil, was shown to exert less potent calcium channel blocking activity and lower cardiotoxicity compared to S-isomer [107, 108] while maintaining its ability to reduce doxorubicin chemoresistance in the same extent as its racemate [109]. Another second-generation P-gp inhibitor PSC833, also known as valspodar, is a 10–20-fold more potent analog of cyclosporine D, but contrary to its parent compound, valspodar does not exert immunosuppressive activity. However, these compounds showed to be potent CYP 3A4 enzyme inhibitors. Therefore, significant undesirable pharmacokinetic interactions between anticancer drugs were observed, that result in delayed elimination and increased toxicity [110,111,112,113]. It is because many anticancer agents, such as doxorubicin, paclitaxel, and vinblastine, are metabolized by CYP 3A4 enzyme.

Aforesaid limitations encouraged the development of the third-generation P-gp inhibitors, that possess low toxicity, higher specificity, and binding affinity to P-gp and do not interact with the CYP450 3A4 enzyme. These inhibitors include tariquidar, zosuiqudar, elacridar, biricodar, and ONT-093. Design of these novel compounds was based on the structure-activity relationship studies. Tariquidar is a derivative of anthranilic acid. It has 4-fold higher affinity for P-gp than vinblastine, and 20-fold higher affinity than paclitaxel [114]. Tariquidar is a non-competitive P-gp inhibitor [114]. Phase I trial showed that tariquidar is well tolerated when combined with doxorubicin, docetaxel, or vinorelbine [115]. However, two phase III clinical trials of tariquidar in combination with paclitaxel plus carboplatin or vinorelbine alone for non-small cell lung cancer were discontinued. These decisions have been made due to high levels of toxicity observed in the tariquidar arms (QLT Inc. Form 8-K). Another P-gp inhibitor biricodar showed acceptable levels toxicity and good tolerability [116], but was not very efficient [117].

Due to unsuccessful results of the third-generation inhibitors in clinical trials, screening of natural substances has been started. These plant-based compounds belong to the fourth-generation P-gp inhibitors and include alkaloids, terpenoids, flavonoids, coumarins, and saponins. It has long been known that grapefruit juice induces P-gp-related drug efflux [118]. In a recent study by Zhang et al., incubation of doxorubicin-resistant osteosarcoma cells with resveratrol for 48 h caused an almost 7-fold decrease of doxorubicin antiproliferative activity when compared to cells incubated with doxorubicin alone. Also, an increase in intracellular concentration of drug and downregulation of MDR1/P-gp gene expression was determined [119]. Another natural P-gp inhibitor, citrus methoxyflavone nobiletin, was found to inhibit P-gp efflux function and increase the efficacy of paclitaxel, doxorubicin, docetaxel, and daunorubicin in ovarian cancer and colorectal adenocarcinoma cells [120]. Moreover, it was shown that flavonoids inhibit not only P-gp but also BCRP, thus increasing an intracellular concentration of anticancer compounds that are BCRP substrates [121].

BCRP inhibitors

BCRP is the most recently found ABC transporter. The substrates of BCRP possess several common structural features such as a planar structure, hydrophobic regions, aromatic systems, 7 to 20 carbon atoms and oxygen-containing groups [136]. There are only few selective BCRP substrates. Usually, the substrates of BCRP have the high affinity to P-gp, as well. One of the first discovered BCRP inhibitors was fumitremorgin C, an indole alkaloid isolated from Aspergillus fumigatus. According to in vitro studies, it is a very effective BCRP inhibitor that almost completely reverses resistance to mitoxantrone, doxorubicin, topotecan and paclitaxel in BCRP overexpressing MCF-7 breast cancer cells [137]. Similar results were found in colon carcinoma cells S1-M1-3.2 that expressed low levels of P-gp and MRP [138].

However, fumitremorgin C has been reported to cause severe neurotoxicity, which impeded its application in clinical practice. Therefore, its nontoxic analog Ko143 was developed. It is a very potent BCRP inhibitor that exerts its effect at nanomolar concentrations [139]. Recently, it was found that at concentrations higher than 1 μM Ko143 inhibits P-gp and MRP-1 [132]. This inhibitory effect on all three ABC transporters may be favorable in order to increase the efficacy of anticancer therapy but may also increase the risk of toxicity.

MRP-1 inhibitors

MRP-1 is another transmembrane protein that belongs to the ABC transporter family and pumps drug substances out of the cell. It was first discovered in human small cell lung carcinoma cells in 1992 [77]. As previously described P-gp and BCRP, MRP-1 is also overexpressed in a wide variety of solid tumors and it is considered to be a negative prognostic factor of the disease. This protein takes part in the efflux of well-known common anticancer drugs such as doxorubicin, vinblastine, methotrexate, and recently developed compounds, for instance, tyrosine kinase inhibitors. Numerous in vitro and in vivo studies have shown that inhibition of MRP-1 or downregulation of its gene expression leads to chemosensitization of cancer cells to various anticancer agents [140,141,142]. One of the most potent MRP-1 inhibitors is a pyrazolopyrimidine derivative reversan. Reversan also inhibits P-gp, and it showed favorable toxicity profiles in murine models [143]. Regardless of favorable results in vitro or in mice, no clinical trials were conducted with MRP-1 inhibitors yet.

Physical methods

Besides inhibition of pH-regulating proteins or ABC transporters with chemical compounds, physical methods can also be used to improve drug delivery into cancer cells. These methods include sonoporation by low-intensity ultrasound and electroporation.

Sonoporation

In late 1940s, ultrasound was first applied for medicinal diagnostics [144]. In recent decades, there is an increasing interest by scientists in the application of ultrasound in anticancer therapy. There are numerous studies proving that ultrasound combined with microbubbles may enhance anticancer drug delivery into tumor cells [145,146,147].

Microbubbles consist of hydrophobic, usually fluorinated gas coated with 10–100 nm layer made of polymers, proteins, and lipids. In order to increase the specificity of microbubbles to tumor tissues, a particular ligand specific to the cell surface receptors can be attached to them. Due to the acoustic pressure of ultrasound, microbubbles start to shrink and expand periodically. This process is called cavitation. When the acoustic pressure reaches a certain threshold, a collapse of microbubbles occurs [148]. It is thought that cavitation or explosion of microbubbles creates temporary pores in the cell membrane through which the drug passively enters the cells (Fig. 3) [149,150,151]. These pores close up as soon as ultrasound exposure is terminated [149, 151]. The drug solution can be either mixed with microbubbles or added before.

Fig. 3
figure 3

Sonoporation-induced formation of pores in the cell membrane. Ultrasound waves cause contraction, expansion, and explosion of microbubbles. This process leads to the rupture of membrane and temporary pores formation

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According to some studies, ultrasound may improve not only passive diffusion but also the active transport of drugs. It is believed that ultrasound may cause changes of ion channels; therefore, intracellular Ca2+ concentration increases and causes cytoskeletal rearrangement [152]. These processes stimulate endocytosis and drug delivery into cells.

In order to cause microbubble cavitation, low-frequency (0.4–3.0 MHz) ultrasound is used and duty cycle may vary from less than 1 to 90%. Long duty cycle and high intensity of ultrasound may cause tissue damage [153].

Results from in vitro studies confirm the possible benefit of ultrasound application in chemotherapy. Escoffre et al. determined that ultrasound and microbubbles combination increased doxorubicin antiproliferative activity by 2.5-fold [145]. Similar results were published by Piron et al. [146]. Grainger et al. investigated the effect of ultrasound on drug delivery in 3D cancer cell cultures. It was shown that ultrasound when used in combination with microbubbles increases nanoparticle penetration into breast cancer cell spheroids [147]. There is a lack of clinical trials, though. There is only one phase I clinical trial that showed promising results on the application of ultrasound and microbubbles against pancreatic cancer. Ultrasound prolonged survival from 8.9 to 17.6 months when compared to control and did not increase drug toxicity. However, patient cohort was too small (n = 10) to make reliable conclusions [154]. In order to evaluate the impact of ultrasound on the efficacy of chemotherapy, further in vivo studies and clinical trials are needed.

Electroporation

Electroporation, also known as electropermeabilization, technique is similar to sonoporation. The main difference is that instead of ultrasound the cells are exposed with short pulses of high voltage (usually 0.5–1 V) electrical field [155]. Electric field causes structural the rearrangement of lipid molecules of the cell membrane. This results in creation of hydrophilic pores and increased permeability of the cell membrane. Electroporation can be reversible or irreversible. The pores created by reversible electroporation are temporary and remain at least several minutes, depending on their size and duration of electric pulse [156]. In contrary, during irreversible electroporation, certain threshold of the strength and duration of electrical pulse is exceeded and cell death is caused. There are many in vitro and in vivo [157,158,159] studies that demonstrate an increased efficacy of anticancer drugs, such as bleomycin, gemcitabine, or cisplatin, when combined with electric pulses [160,161,162]. In phase II clinical trial (n = 52), the influence of electroporation on the efficacy of bleomycin in treatment of superficial metastasis of various cancers was tested. One month after the first application of electroporation, the reduction in tumor size was observed in 50 of 52 patients. After the second course of electroporation, 18 of 27 patients had partial or complete response. One hundred sixty-nine of 257 tumor nodules disappeared and in 89 size reduction was determined [163]. In contrary to sonoporation, electrochemotherapy is currently used in clinical practice as palliative treatment in case of melanoma [164], basal and squamous skin cancer [165], and skin metastasis from tumors of non-skin origin [166]. Many researchers are still working in the field of electrochemotherapy in order to investigate the possible application of the method on other types of cancer, such as bladder [167] or esophageal cancer [168].

Nanocarrier systems

To increase drug delivery to tumor cells and to reduce toxicity against normal tissues, various targeted nanocarrier systems are being developed. They include micelles, liposomes, dendrimers, gold nanoparticles, mesoporous silica nanoparticles, superparamagnetic iron oxide nanoparticles, carbon nanotubes, and quantum dots [169]. Non-targeted nanoparticles accumulate in tumor tissues due to leaky and defective blood vessels, and reduced lymphatic drainage [170]. Targeted nanoparticles bear particular ligands that have high binding affinity to cancer cell surface molecules [171].

Nanoparticles carry an anticancer agent to the target site, where drug can be released due to various stimuli, such as pH changes, reduction reactions caused by glutathione sulfhydryl groups, enzymatic activity, magnetic or electric field, ultrasound [169]. Recently, mono-allyloxylated cucurbit[7]uril (AO1CB [7]) nanovesicles have been created [172]. It works as a nanocontainer for various drugs and proteins. When these vesicles are affected with UV light or near-infrared two-photon laser, the allyloxy tails of (AO1CB [7]) react with glutathione or other intracellular molecule containing thiol group, thus resulting in targeted drug delivery. Although this method showed to increase doxorubicin delivery into cervical cancer cells, its efficacy needs to be investigated in further in vitro and in vivo studies.

For stronger therapeutic efficacy, various transport modulators can be incorporated into these systems. Janoniene et al. combined porous silicon nanoformulations with transport modulator—selective carbonic anhydrase IX inhibitor [61]. Porous silicon nanoparticles loaded with doxorubicin and conjugated with carbonic anhydrase IX inhibitor enhanced target drug delivery towards tumor tissues and improved doxorubicin penetration into cancer cells. Carbonic anhydrase IX inhibitor also enhanced drug loading efficacy into nanoparticles and improved drug release profile by reducing the premature release of doxorubicin.

Dual delivery systems consisting of an anticancer drug and nucleic acids, that silence the expression of drug efflux transporters genes, are being developed, as well. Meng et al. showed that co-delivery of doxorubicin and siRNA, that knocks down the expression of P-gp, by mesoporous silica nanoparticles increased intracellular delivery and cytotoxicity of doxorubicin [173]. Silencing of P-gp gene leads to a reduction of drug efflux and a decrease in pump-mediated drug resistance.

At this point, there are 49 clinical trials on the field of cancer with a term “nano” listed on ClinicalTrials.gov database and most of them are still recruiting or ongoing. Two liposomal drugs—doxorubicin (Doxil®) and irinotecan (Onivyde®), one polymeric nanoparticle drug—leuprolide acetate (Eligard®), and one protein nanoparticle drug—albumin-bound paclitaxel (Abraxane®)—are currently approved for clinical use. Although, simple nanoparticles show higher efficacy and reduced toxicity, progressive trends towards more complex nanomedicine and dual delivery systems can be seen in the research field.

Conclusions

Despite various attempts to reverse MDR, it still remains one of the most important problems of chemotherapy. So far, neither pH modulators nor ABC transporter inhibitors or application of ultrasound is applied in clinical practice. However, novel V-ATPase or ABC-transporter inhibitors, especially third- and fourth-generation P-gp inhibitors (such as zosuiqudar, elacridar and resveratrol), show good efficacy in in vitro and in vivo models. CA and NHE-1 inhibitors or ultrasound in combination with microbubbles also demonstrate promising results in modulation of anticancer drug penetration. Currently, there are two ongoing clinical trials (NCT03458975 and NCT02233205) investigating the efficacy of sonoporation on the delivery of anticancer drugs, but the results are not published, yet. Instead of microbubbles, researchers are developing nanobubbles as they are smaller and can easier penetrate through blood vessels [174]. Electrochemotherapy is approved for the treatment of a few certain types of cancers, but researchers are still trying to adapt the method for the treatment of cancer in body cavities. Besides physical methods, various nanocarrier systems are gaining a great attention in an anticancer therapy, as well. Incorporation of transport modulators into these delivery systems is an unsaturated research niche area warrant for further investigation.