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Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
CHAPTER 22
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles Introduction Extracellular acidity is a constant finding in all kind of cancers and its causes have been extensively discussed in Chapters 2–4. It represents an important instrument in the matrix degradation/migration/invasion process and at the same time a problem and an opportunity for the oncologist. The opportunity stems from the fact that this phenomenon is mainly restricted to malignant tissues. Nano-systems, especially those that are activated by pH, makes it possible to target cancer while preserving normal cells and permitting the delivery of drugs directly to the tumor. At the same time, these systems can by-pass biological barriers and avoid the extrusion capabilities of multidrug resistance (MDR). The extracellular acidity of tumors represents the perfect opportunity for the development of pH-sensitive nanoparticules (NPs) that release the drug in the extracellular substance of the tumor or directly inside the malignant cells.1
The problems of chemotherapy Among the many problems of chemotherapy, there are three that we shall discuss here: (1) The bioavailability of the chemo drug at the tumor site; (2) An adequate penetration into the malignant cell; (3) Toxicity to normal cells.
The bioavailability of the chemo drug (CD) at the tumor site Once injected or ingested, the drug enters the circulatory system where it is distributed throughout the entire organism. For this, the drug must be hydrosoluble in order to be transported by the blood.2 For example, curcumin a very interesting nutraceutical for cancer treatment is not soluble in any kind of non-toxic solvents and thus, it cannot be used as a therapeutic substance. Once in the blood, the CD may be denaturalized by normal blood components or may be bound to proteins that decrease its activity. The goal of CDs is to achieve the highest possible concentration at the tumor site and the lowest possible concentration at normal tissues. If the drug can be “protected” from An Innovative Approach to Understanding and Treating Cancer: Targeting pH https://doi.org/10.1016/B978-0-12-819059-3.00022-8
© 2020 Elsevier Inc. All rights reserved.
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destruction (by macrophages) or inactivation (by other substances) during its widespread distribution throughout the organism, a higher concentration of active drug can be achieved at the required site of action. Nanoparticles (NPs) attached to CDs or CDs inside NPs can do this job. Another objective of treatment is the intracellular acidification of malignant cells with the goal of inducing acid stress and apoptosis, certain specialized NPs can do this too. But, how does this NP-CD complex know where to release the CD? This is the point where pH plays the capital role: NPs can be constructed in such a way that they release the drug specifically in sites with low pH, such as the extracellular space (ECS) of tumors. This are called the pH-sensitive NPs.
An adequate penetration into the malignant cell The NP “protects” the drug and releases it specifically at the tumor’s ECS. A second problem may ensue now: the acidic pH may inactivate the CD. Extracellular acidity has been found to be an important factor of chemoresistance. This is particularly evident in weakly basic anti-cancer drugs.3,4 Proton pump inhibitors5,6 and buffers like bicarbonate7 were proposed to modify this situation. But the relation between chemoresistance and pH is not limited to the extracellular acidity of tumors: the acidity of intracellular vesicles may play a role too. One of the mechanisms used by cancer cells to decrease the activity of chemotherapeutic drugs consists in sequestering them in endocytic vesicles.8 With certain CDs (e.g., doxorubicin) is not enough to carry them to the specific site of action, it is also necessary to avoid sequestration by protonation due to the acidic conditions of the tumor’s ECS. Using doxorubicin (DOX), Mellor and Calaghan found that P-gp (P-glycoprotein) and extracellular acidity represented similar and equivalent problems for an adequate anti-cancer effect. The effects of doxorubicin were limited to the outer layer of tumor cells and failed to reach the more hypoxic areas.9,10 Daunorubicin, doxorubicin and mitoxantrone are weak bases and therefore activity in a highly acidic medium is substantially reduced. Paclitaxel on the other hand is not affected by pH. Extracellular acidity is highly heterogeneous, with very acid areas coexisting with less acidic ones. This means that acid-sensitive chemotherapeutic drugs will achieve good results in less acidic areas, but will fail in the more acidic ones. The very acidic niches that remain unharmed by the treatment will become the core for relapse and/or clonal evolution toward resistance. Here again, pH-sensitive NPs can do the job by facilitating penetration into the cell and releasing the CD inside the cell. In this case, the design of the NP is different from the first situation.
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
Up to now, we have two different kinds of NPs: (a) NPs that release the CD in acidic sites, and (b) NPs that penetrate the malignant cell with its cargo releasing the drug inside the cell. There are also five other types of NPs in which we are interested: (c) an NP that can acidify the cytoplasm; (d) an NP that deposits its load to the cell surface but does not let it enter to the cell; (e) the double releasing NP; (f) the multidrug NP; (g) the multitarget NP (Fig. 1).
Fig. 1 Different types of NPs related to the pH gradient inversion: (1) pH-sensitive NP that releases the drug in acidic extracellular matrix; (2) pH-sensitive NP that facilitates drug penetration inside the cell; (3) pH-sensitive NP that acidifies the intracellular milieu; (4) pH-sensitive NP that deposits the drug on the cell surface but does not allow drug penetration inside the cell.
Toxicity to normal cells The above mentioned NPs are designed in such a way that drug release mainly takes place in the malignant tissues and by-passing the normal ones. And this is possible due to cancer’s altered pH homeostasis. In this chapter we will focus on these four types of pH-sensitive NPs: the ones that release the drug in acidic sites, the NPs that release the drug inside the malignant cell, the NPs that acidify the intracellular compartment and the NPs that concentrate the drug on the cell surface without allowing penetration. We will also touch briefly the other types of NPs.
pH-sensitive NPs that release the drug in acidic sites Many different nanocarriers have been tested in combination with chemotherapeutic drugs (CDs). These nanoparticles (NPs) are expected to increase the efficacy of
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therapeutic agents while reducing unwanted side effects. There are NPs that have been designed considering the advantage of the low pH of the extracellular space in tumors, being activated by this low pH and releasing the chemotherapeutic drug. This leads to a high accumulation of the drug at the tumor site.11 Hruby et al.12 developed a polymeric micellar system for the delivery of doxorubicin that released 43% of the drug at pH 5 and only 16% at pH 7.4. Meng et al.13 created a mesoporous silica NP for doxorubicin release at low pH. Many of these pH-activated delivery systems are engineered in such a way that they can reach lysosomes and nuclei, and be selectively released in the tumoral extracellular matrix. Pegylated liposomal doxorubicin is doxorubicin protected by a liposomal layer reducing the uptake by the reticulo-endothelial system and stably retaining the drug as a result of liposomal entrapment. These features increase its permanence in circulation, leading to an increased tumor uptake.14 It is not pH-activated, but it is a proof of concept that better vehicles for chemotherapeutic drugs can succeed in achieving improved results. In order to produce a maximal tumor concentration of a cationic drug such as doxorubicin (Dox) a specific nanocarrier must be engineered. Dox enters the cell through diffusion and interferes with the topoisomerase II-DNA complex leading to cell death.15 The idea of delivering NPs capable of transporting Dox unchanged at normal pH to sites were pH is lower like the tumor extracellular matrix, where the drug is released of this protection, is particularly attractive and it has been the object of great amount of research. The pH-activated drug release NPs are all based on the particular acid conditions of the pHe.16 Box 1 and Fig. 2.
BOX 1 pH-sensitive NPs must fulfill three criteria (1) Stability at physiologic pH. (2) Destabilization/degradation or conformational changes at pH <6.8. (3) Ability to release their load when pH falls below the trigger pH.
Fig. 3 shows the building of a pH-sensitive NP. Changes in pH introduce conformational changes in the NP that lead to disintegration or degradation of the NP thus releasing the drug.
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
Fig. 2 The pH-sensitive NP protects the drug while in the circulatory system. When it reaches an area with low pH, such as in tumors, it releases the drug. There is no drug release in normal tissues because their extracellular pH is markedly higher than in tumors. The small yellow circles in the blood vessel represent possible substances that can decrease the chemotherapeutic activity. The NP “protects” the drug against macrophages, blood proteins, etc.
Fig. 3 Schematic representation of a system of pH-activated drug delivery. The drug is “protected” during its journey through blood vessels and tissues with normal pH. It is released when it encounters an acidic pH.
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pH-sensitive NPs that release the drug inside the cell There are many mechanisms to drive the drug inside the cell. One of them consists in attaching a substance to the NP that has receptors on the cell surface. The substance binds the receptor and the internalization is carried out through the formation of an endosome. The endosomal pH is low and it releases the drug from the nanoparticle. The NP can be a polymer or a micelle charged with the drug, and there is a linker or cleavable bond that attaches the substance to the NP (Fig. 4).
Fig. 4 A mechanism to deliver a chemotherapeutic drug inside the cell. See explanation in the text.
For example, ovarian cancer cells over-express folate receptors in more than 90% of the cases.17 The inhibition of this folate receptors, essential for ovarian cancer metabolism, led to the development of Farletuzumab (a monoclonal antibody against folate receptor alpha). It failed in phase III clinical trials for relapsed ovarian cancer, but showed some minor benefits.18 An NP loaded with a chemotherapeutic drug such as a vinca alkaloid can be bound either to farletuzumab or to folate through a cleavable linker (Fig. 4, panel 1). This NP Vinca-Folate or Vinca-Farletuzumab targets the folate receptor at the cell membrane (Fig. 4, panels 2 and 3) and induces the internalization of the complex through the formation of an endosome (Fig. 4, panels 4 and 5). The acidic condition of the endosome (pH 5) breaks the cleavable linker and releases the drug (Fig. 4, panel 6).
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
Finally, endolysis (panel 7) releases the Vinca alkaloid in the cytoplasm where it acts against the microtubular system.19 Vintafolide or EC145 is the name of an experimental product fabricated on these parameters. Actually, this compound does not incorporate the vinca alkaloid into a micelle. It is directly attached to the pH-sensitive linker. It is in phase II Clinical Trial.20 In a phase I clinical trial Vintafolide showed low toxicity for the patients and very few side effects. Incorporating a micelle to the structure of Vintafolin, as seen in Fig. 4 would allow the internalization of more than one drug simultaneously. A step forward is the development of dual pH-activated nanoparticles, like the one proposed by Du et al.21 This dual pH system has the ability of reversing the surface charge of the particle when meeting the acidic tumor environment. In this manner the internalization of the particle is facilitated. When the particle inside the cell enters the endosomes, which are even more acidic than the extracellular environment, doxorubicin is further released. These dual systems protect the drug from the extracellular acidity of tumors, and facilitate its penetration inside the cell (Fig. 5).
Fig. 5 A dual pH-sensitive NP. (1) The NP-drug complex has a negative charge while circulating in the blood vessels. (2) When it leaves the blood vessels at the tumor site, the acidity of the tumoral extracellular matrix produces an electrical inversion that facilitates the penetration (3)–(5) into the cell. The extreme acidity of the endosomes further releases the drug (6) and (7).
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The intracellular delivery of substances by pH-activated nanoparticles is not exclusive for drugs, they can also be used to deliver other anti-cancer tools such as SiRNAs.22 Fig. 6
Fig. 6 A double coated NP delivery system for siRNA. (1) The first pH-sensitive coat drives the NP to the tumor’s ECS where it loses its first coat. The second coat is revealed with a strong negative electrostatic charge that increases the cellular uptake. (2) Inside the cell, the second coat is shedded and the siRNA is released.
shows a delivery system that transports siRNA instead of drugs. It has a double coating and loses the first coat when it arrives to a low pH region. At this point the negative charge of the second coat facilitates NP-siRNA’s entry to the cytoplasm. Then, the siRNA is released.
NPs that can acidify the cytoplasm It has been established that cytoplasmic acidification is the main objective for the reversal of pH alterations in cancer. Carbone et al.23 developed an NP in which they synthesized a chemical cage24 that shields a proton (H+) and coupled it to a gold nanoparticle. When this AuNP-caged H+ is taken up by the cell, stimulation with ultraviolet light releases the proton from the cagea and the freed H+ combines with CO3H producing carbonic acid that decreases intracellular pH (Fig. 7). a
“The idea behind the caging technique is that a molecule of interest can be rendered biologically inert (or caged) by chemical modification with a photoremovable protecting group”. From Ellis Davies.24
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
Fig. 7 (1) The NP consists of a chemically caged proton bound to a gold particle. (2) and (3) The NP is internalized into the cell. (4) Ultraviolet light stimulation “breaks” the cage releasing the proton that acidifies the cytoplasm.
These type of NPs have one drawback: their use would be limited to those tissues that can be irradiated with ultraviolet light. In the future, these NPs may become useful for treating oral cavity and oropharynx tumors. We also believe that it would be perfectly feasible to create endoscopes that can deliver UV light inside cavities.
NPs that deposit their load on the cell surface but do not let it enter the cell Inhibition of carbonic anhydrase (CA) is an important step in the reversal of the inverted pH gradient. The problem is that CA is a family of different isoforms. Those related to cancer are the membrane CAs: CAIX and CAXII. The model inhibitor of CAs is acetazolamide that works as a pan-inhibitor. This means that acetazolamide inhibits all the isoforms of CA, including cytoplasmic and membranous CAs. CAs are housekeeping enzymes and therefore fully inhibiting them all would be highly toxic. A drug delivery system able to inhibit only the membranous CAs of tumors without affecting normal cells would be the goal.25 There is advanced research with CA inhibitors attached to an NP that impedes cell penetration.26,27 These mechanisms would preserve intracellular CAs and deliver the drug mainly to malignant tissues that over-express membrane CAs. Fig. 8.
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Fig. 8 A non-permeable carrier transports the carbonic anhydrase inhibitor to the cell surface.
Double release NPs Fig. 5 is an example of a double release NP. When the NP reaches the tumor’s ECS undergoes a change in polarity that increases its penetration into the cell. Inside the endosome, the very low pH induces an NP’s conformational change with further release of the drug to the cytoplasm. This is just one example of many. Miao et al. developed a similar dual NP loaded with DOX.28 Kim et al.29 synthesized an interesting system of double targeting micelles transporting doxorubicin (DOX). In the first step these micelles protected DOX and facilitated its arrival to the cell surface. Then a second component of the micelle containing folate engaged the folate receptors of the cell membrane inducing endocytosis. The ensuing endocytolysis, released the drug. This system could be applied successfully to cells with the MDR phenotype. Actually this is a system of double targeting more than double release.
Multidrug release NPs NPs with the ability to deliver more than one drug have been developed recently.30 Liao et al.31 fabricated a system that delivers three chemotherapeutic drugs simultaneously:
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
doxorubicin, camptothecin and cisplatin. In this case the mechanism of release was not through pH-sensitive NPs but through light stimulation. In theory, a similar system could be developed with a pH-triggered release which would make the compound more focused on cancer cells.
Radiosensitizing NPs Ma et al.32 developed a NP based on the delivery of a carbonic anhydrase inhibitor and quercetin. This construction decreased the effects of hypoxia on tumors increasing the effects of radiotherapy. NP binding carbonic anhydrase is an interesting concept to deliver drug into hypoxic areas of the tumor.33
The making of an NP A useful pH-sensitive NP for oncological use must combine at least two basic features: (1) Ability to store the drug in stable conditions at normal pH. (2) Release the drug when the pH decreases below a pre-established threshold. These can be accomplished by adding chemical groups to the nanomaterial with the ability to: (a) accept or donate protons; (b) produce physical changes according to the pH. Polymer-based drug delivery Biopolymers (BPs) are the most frequently used nanocarriers for drug delivery. These compounds have some features that make them the best choice in the case of pHsensitive systems: (1) BPs are biodegradable. (2) BPs are compatible with human tissues and body fluids. (3) BPs are easy to manipulate in the laboratory.34 (4) BPs have the ability to respond to ionization with conformational changes. Most of the pH-sensitive NPs are actually pH-sensitive polymers that can respond with changes in their physical properties when they encounter changes in the pH of their environment. In many of these NPs the polymer is dissolved when subjected to certain pH changes.35 This is the case of NPs made of poly(β-amino) ester shown in Fig. 9.36 These polymers can transport the drug in two different ways37: (1) the drug is physically entrapped in the NP; (2) The drug is covalently bound to the NP (Fig. 10). In the second case the drug is bound to the surface of a nanocarrier through a covalent acid-labile bond. The chemical bond is hydrolyzed when the pH decreases, releasing the drug.
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Fig. 9 Degradation or disassembly of the NP releases the drug when pH is lowered from the physiologic to the acidic range.
Micelle-based drug delivery Another example of disassembly induced by low pH is the fabrication of NPs formed by micelles that are bound to hydrophobic drugs by a linker. The acidic pH dissolves the micelles releasing the drug (Fig. 10).
Fig. 10 Upper panel: two different mechanisms of drug transport by a polymer: inside the nanocarriers (left) or attached through a chemically labile bond (the linker) (right) Lower panel: a pH-sensitive micellar NP.
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
Polyester based NPs release acidic degradation products. This may represent a drawback if the degradation products remain in the ECS increasing its acidity. Hefferman et al.38 developed an NP based on a new hydrophobic polymer called polyketal that avoids the problem. The possibilities for the development of new site-specific pH-sensitive NPs for the delivery of drugs and other types of molecules are enormous and we are just at the beginning. Table 1 is only a small sample of pH-sensitive NPs.
Table 1 A summary of pH activated drug delivery systems for cancer treatment Reference
Yang et al. (2018)
Mechanism 39
Ju et al. (2017)40
Ehi-Eromesele et al. (2017)41 Guo et al. (2017)42 Wu et al. (2017)43
Li et al. (2016)44
Jiang et al. (2017)45
Piao et al. (2017)46 Gisbert-Garzaran et al. (2017)47 Zignani et al. (2000)48
Poly-L-lysine-lipoic acid modified by dimethylmaleic anhydride used as a carrier for doxorubicin with enhanced cell internalization and intracellular pH release. A liposome prepared with a synthetic oligopeptide lipid, soy phosphatidylcholine, and cholesterol showed that this pH sensitive drug carrier enhanced tumor cell uptake and increase cytoplasmic distribution. Magnetic nanoparticles coated with silica, pH sensitive particles were synthesized as carriers of 5 FU. At pH 5 this NP released three times more 5 FU than at pH 7.4. A gene transfection system was engineered in which the gene is part of a pH-responsive charge-convertible ternary complex. A graft copolymer of poly(butylenes succinate)-g-cysteamine-bi-poly (ethylene glycol) was synthesized for doxorubicin transport. This micellar system could switch from less than 25% to 84% of doxorubicin release according to the media pH. A dual sensitive biodegradable micelle for drug transport was built with maximal release of the drug inside the tumor cell. The micelle was sensitive to pH and redox conditions. The micelles were quickly and fully internalized by cellular endosomes. A transactivator of transcription (TAT) nanoparticle was developed with increased intracellular retention and nuclear translocation of a platinum based chemotherapeutic drug. The intranuclear acid conditions triggered the TAT activity (not the extracellular acidity). Gold nanocages with zwitterionic pH sensitive coating were used to increase delivery of chemotherapeutic drugs to the tumor. pH-responsive mesoporous of silica and carbon was used to increase delivery of chemotherapeutic drugs into tumors. pH-sensitive release of drug was achieved with egg phosphatidylcholine liposomes adding N-isopropylacrylamide (NIPA) copolymers.
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Clinical implications Chemotherapeutic drugs are generally very toxic. Chemotherapy can be defined as a “controlled intoxication” where art and science join to keep the drug within a narrow therapeutic range. If that range is exceeded the patient will suffer serious adverse effects and if the range is not achieved the treatment will be ineffective. Therefore, the way of administering the drug (drug delivery system) is of capital importance.49 The other main concern in oncology is to deliver the highest possible amount of drug to the tumor and ideally none to the rest of the organism. Nano-systems that are pH-dependent will be a part of the new delivery systems that may eventually change oncological practice as we know it today. Liu et al.1 wrote: “Stimulus-responsive nanomaterials are termed as ‘smart’, ‘intelligent’, or ‘environmentally sensitive’, and have greater potential than traditional delivery systems.” A few interesting objectives can be achieved by using pH-sensitive nanocarriers: (1) Specific and focalized delivery of drugs to the tumor without toxicity to normal cells. (2) Intracellular acidification limited to cancer cells. (3) Counteracting or by-passing multidrug resistance (MDR). (4) Intracellular delivery of siRNAs that can block oncogenic pathways and proteins. (5) Increased stability and permanency of the drug in circulation. (6) Higher concentrations of the drug without generating intolerable toxicity. (7) Increased retention of the drug inside the tumor. These compounds are not yet in clinical use, but they will be soon. The hand-foot syndrome (palmar-plantar erythrodysesthesia) is a well-known adverse event with some cytotoxic CDs50,51 including the non pH-sensitive pegylated liposomal doxorubicin (PLD). The incidence with PLD is close to 50%.52 This problem results in treatment interruption in many cases. However, it is less frequent when DOX is used alone.53 This led to the conclusion that prolonged circulation due to the pegylated liposome NP increased leakage to non tumor-specific sites.54 As a speculation, we may assume that using pH-sensitive NPs instead of PLD, the leakage probably would not be avoided. However the leaked NPs would not release DOX and therefore a foot and hand syndrome would be prevented. The advantages of a pH-sensitive NP chemotherapy drug delivery become very evident with the example described above.
Conclusions We have oversimplified this chapter, sacrificing scientific details for the sake of clarity to offer a better understanding of this complex topic. The first nanoparticles (NPs) were mainly designed for drug delivery improving its pharmacokinetics, prolonging its presence in the circulatory system and decreasing its
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
degradation or inactivation before reaching its target. Then, new and more ambitious objectives were set for NPs: more focused delivery to the tumor, multidrug delivery, siRNA delivery, specific intracellular targeting, controlled drug release, environmental sensitive response, etc. After the success of the pegylated liposomal doxorubicin (PLD), that has been used intensively in the last 10 years, a considerable effort has been dedicated to pH-sensitive drug carriers and very interesting developments have been achieved. The pH paradigm of cancer is a targetable soft spot of cancer that exposes malignant cells to an easier attack by pH-sensitive nanocarriers. However, none of these new pH-sensitive carrier systems have yet been approved for human use. It is highly possible that we shall see breakthroughs on this issue in the near future. It opens the possibility to target certain components of the pH paradigm that, at present, lack efficient blockers. This would be the case of NBC (sodium/bicarbonate cotransporter), just to mention one example. As a membrane protein we think it may be targeted with nanocarriers that can protect normal cells from the attack. The many broad and interesting possibilities that nanocarriers can offer to the world of oncology have not yet been fully explored. We trust that these tools will become part of clinical oncology very soon. This chapter is only a brief glance at this subject that would require—and deserves—many dedicated volumes. Peer et al.55 wrote: “Nanotechnology has the potential to revolutionize cancer diagnosis and therapy. Advances in protein engineering and materials science have contributed to novel nanoscale targeting approaches that may bring new hope to cancer patients.” We share this concept.
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10. Mahoney BP, Raghunand N, Baggett B, Gillies RJ. Tumor acidity, ion trapping and chemotherapeutics: I. Acid pH affects the distribution of chemotherapeutic agents in vitro. Biochem Pharmacol 2003; 66(7):1207–18. 11. Lim EK, Huh YM, Yang J, Lee K, Suh JS, Haam S. pH-triggered drug-releasing magnetic nanoparticles for cancer therapy guided by molecular imaging by MRI. Adv Mater 2011;23(21):2436–42. Ulbrich K. Polymeric micellar pH-sensitive drug delivery system for doxorubicin. J 12. Hruby´ M, Konˇa´k C, Control Release 2005;103(1):137–48. 13. Meng H, Xue M, Xia T, Zhao YL, Tamanoi F, Stoddart JF, et al. Autonomous in vitro anticancer drug release from mesoporous silica nanoparticles by pH-sensitive nanovalves. J Am Chem Soc 2010; 132(36):12690–7. 14. Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated liposomal doxorubicin. Clin Pharmacokinet 2003;42(5):419–36. 15. Tacar O, Sriamornsak P, Dass CR. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J Pharm Pharmacol 2013;65:157–70. 16. Du JZ, Mao CQ, Yuan YY, Yang XZ, Wang J. Tumor extracellular acidity-activated nanoparticles as drug delivery systems for enhanced cancer therapy. Biotechnol Adv 2014;32(4):789–803. 17. Armstrong DK, White AJ, Weil SC, Phillips M, Coleman RL. Farletuzumab (a monoclonal antibody against folate receptor alpha) in relapsed platinum-sensitive ovarian cancer. Gynecol Oncol 2013; 129(3):452–8. 18. Vergote I, Armstrong D, Scambia G, Teneriello M, Sehouli J, Schweizer C, et al. A randomized, double-blind, placebo-controlled, phase III study to assess efficacy and safety of weekly farletuzumab in combination with carboplatin and taxane in patients with ovarian cancer in first platinum-sensitive relapse. J Clin Oncol 2016;34(19):2271–8. 19. Marchetti C, Palaia I, Giorgini M, De Medici C, Iadarola R, Vertechy L, et al. Targeted drug delivery via folate receptors in recurrent ovarian cancer: a review. Onco Targets Ther 2014;7:1223. 20. Sharp M, Corp D. Study of EC145 in patients with advanced ovarian and endometrial cancers. In: ClinicalTrials. gov. Bethesda, MD: US National Library of Medicine; 2013. Available from:http://clinicaltrials.gov/show/ NCT00507741. [NLM identifier: NCT00507741]. 21. Du JZ, Du XJ, Mao CQ, Wang J. Tailor-made dual pH-sensitive polymer–doxorubicin nanoparticles for efficient anticancer drug delivery. J Am Chem Soc 2011;133(44):17560–3. 22. Sun CY, Shen S, Xu CF, Li HJ, Liu Y, Cao ZT, et al. Tumor acidity-sensitive polymeric vector for active targeted siRNA delivery. J Am Chem Soc 2015;137(48):15217–24. 23. Carbone M, Sabbatella G, Antonaroli S, Remita H, Orlando V, Biagioni S, et al. Exogenous control over intracellular acidification: enhancement via proton caged compounds coupled to gold nanoparticles. Biochim Biophys Acta, Gen Subj 2015;1850(11):2304–7. 24. Ellis-Davies GCR. Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat Methods 2007;4(8):619–28. 25. Liu S, Luo X, Liu S, Xu P, Wang J, Hu Y. Acetazolamide-loaded pH-responsive nanoparticles alleviating tumor acidosis to enhance chemotherapy effects. Macromol Biosci 2019; 19(2):1800366. 26. Stiti M, Cecchi A, Rami M, Abdaoui M, Barragan-Montero V, Scozzafava A, et al. Carbonic anhydrase inhibitor coated gold nanoparticles selectively inhibit the tumor-associated isoform IX over the cytosolic isozymes I and II. J Am Chem Soc 2008;130:16130–1. 27. Sztandera K, Gorzkiewicz M, Klajnert-Maculewicz B. Gold nanoparticles in cancer treatment. Mol Pharm 2018;16(1):1–23. 28. Miao Y, Qiu Y, Yang W, Guo Y, Hou H, Liu Z, et al. Charge reversible and biodegradable nanocarriers showing dual pH-/reduction-sensitive disintegration for rapid site-specific drug delivery. Colloids Surf B: Biointerfaces 2018;169:313–20. 29. Kim D, Lee ES, Oh KT, Gao ZG, Bae YH. Doxorubicin-loaded polymeric micelle overcomes multidrug resistance of cancer by double-targeting folate receptor and early endosomal pH. Small 2008;4:2043–50. 30. Jhaveri A, Deshpande P, Torchilin V. Stimuli-sensitive nanopreparations for combination cancer therapy. J Control Release 2014;190:352–70.
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31. Liao L, Liu J, Dreaden EC, Morton SW, Shopsowitz KE, Hammond PT, et al. A convergent synthetic platform for single-nanoparticle combination cancer therapy: ratiometric loading and controlled release of cisplatin, doxorubicin, and camptothecin. J Am Chem Soc 2014;136(16):5896–9. 32. Ma T, Liu Y, Wu Q, Luo L, Cui Y, Wang X, et al. Quercetin-modified metal–organic frameworks for dual sensitization of radiotherapy in tumor tissues by inhibiting the carbonic anhydrase IX. ACS Nano 2019;13(4):4209–19. 33. Shabana AM, Mondal UK, Alam MR, Spoon T, Ross CA, Madesh M, et al. pH-sensitive multiligand gold nanoplatform targeting carbonic anhydrase IX enhances the delivery of doxorubicin to hypoxic tumor spheroids and overcomes the hypoxia-induced chemoresistance. ACS Appl Mater Interfaces 2018;10(21):17792–808. 34. Prabaharan M, Mano JF. Stimuli-responsive hydrogels based on polysaccharides incorporated with thermo-responsive polymers as novel biomaterials. Macromol Biosci 2006;6:991–1008. 35. Schmaljohann D. Thermo- and pH-responsive polymers in drug delivery. Adv Drug Deliv Rev 2006;58:1655–70. 36. Shenoy D, Little S, Langer R, Amiji M. Poly(ethylene oxide)-modified poly(β-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs: part 2. In vivo distribution and tumor localization studies. Pharm Res 2005;22:2107–14. 37. Rawat M, Singh D, Saraf S, Saraf S. Nanocarriers: promising vehicle for bioactive drugs. Biol Pharm Bull 2006;29:1790–8. 38. Heffernan MJ, Murthy N. Polyketal nanoparticles: a new pH-sensitive biodegradable drug delivery vehicle. Bioconjug Chem 2005;16(6):1340–2. 39. Yang S, Wang Y, Ren Z, Chen M, Chen W, Zhang X. Stepwise pH/reduction-responsive polymeric conjugates for enhanced drug delivery to tumor. Mater Sci Eng 2018;82:234–43. 40. Ju C, Zhang C. Preparation and characterization of pH sensitive drug liposomes. In: Lu WL., Qi XR. (eds) Liposome-Based Drug Delivery Systems. Biomaterial Engineering 2018; Springer, Berlin, Heidelberg. 41. Ehi-Eromosele CO, Ita BI, Iweala EEJ. Silica coated LSMO magnetic nanoparticles for the pHresponsive delivery of 5-fluorouracil anticancer drug. Colloids Surf A: Physicochem Eng Asp 2017;530:164–71. 42. Guo A, Wang Y, Xu S, Zhang X, Li M, Liu Q, et al. Preparation and evaluation of pH-responsive charge-convertible ternary complex FA-PEI-CCA/PEI/DNA with low cytotoxicity and efficient gene delivery. Colloids Surf B: Biointerfaces 2017;152:58–67. 43. Wu S, Zheng L, Li C, Xiao Y, Huo S, Zhang B. Grafted copolymer micelles with pH triggered charge reversibility for efficient doxorubicin delivery. J Polym Sci A: Polym Chem 2017;55(12):2036–46. 44. Li D, Bu Y, Zhang L, Wang X, Yang Y, Zhuang Y, et al. Facile construction of pH-and redoxresponsive micelles from a biodegradable poly (β-hydroxyl amine) for drug delivery. Biomacromolecules 2015;17(1):291–300. 45. Jiang W, Wang J, Yang J, He Z, Hou Z, Luo Y, et al. Acidity-triggered TAT-presenting nanocarriers augment tumor retention and nuclear translocation of drugs. Nano Res 2018;11(10):5716–34. 46. Piao JG, Gao F, Li Y, Yu L, Liu D, Tan ZB, et al. pH-sensitive zwitterionic coating of gold nanocages improves tumor targeting and photothermal treatment efficacy. Nano Res 2018;11(6):3193–204. 47. Gisbert-Garzara´n M, Manzano M, Vallet-Regı´ M. pH-responsive mesoporous silica and carbon nanoparticles for drug delivery. Bioengineering 2017;4(1):3. 48. Zignani M, Drummond DC, Meyer O, Hong K, Leroux JC. In vitro characterization of a novel polymeric-based pH-sensitive liposome system. BBA-Biomembranes 2000;1463:383–94. 49. Langer R. Where a pill won’t reach. Sci Am 2003;288:50–7. 50. Zuehle RL. Erythematous eruption of the palms and soles associated with mitotane therapy. Dermatologia 1974;148:90–2. 51. Nagore E, Insa A, Sanmartin O. Antineoplastic therapy-induced palmar plantar erythrodysesthesia (‘hand-foot’) syndrome: incidence, recognition and management. Am J Clin Dermatol 2000; 1:225–34. 52. Lorusso D, Di Stefano A, Carone V, Fagotti A, Pisconti S, Scambia G. Pegylated liposomal doxorubicinrelated palmar-plantar erythrodysesthesia (‘hand-foot’syndrome). Ann Oncol 2007;18(7):1159–64.
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53. O’Brien ME, Wigler N, Inbar M, Rosso R, Grischke E, Santoro A, et al. CAELYX Breast Cancer Study Group: reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer. Ann Oncol 2004;15(3):440–9. 54. Yokomichi N, Nagasawa T, Coler-Reilly A, Suzuki H, Kubota Y, Yoshioka R, et al. Pathogenesis of hand-foot syndrome induced by PEG-modified liposomal doxorubicin. Hum Cell 2013;26(1):8–18. 55. Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol 2007;2(12):751.
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles Introduction Extracellular acidity is a constant finding in all kind of cancers and its causes have been extensively discussed in Chapters 2–4. It represents an important instrument in the matrix degradation/migration/invasion process and at the same time a problem and an opportunity for the oncologist. The opportunity stems from the fact that this phenomenon is mainly restricted to malignant tissues. Nano-systems, especially those that are activated by pH, makes it possible to target cancer while preserving normal cells and permitting the delivery of drugs directly to the tumor. At the same time, these systems can by-pass biological barriers and avoid the extrusion capabilities of multidrug resistance (MDR). The extracellular acidity of tumors represents the perfect opportunity for the development of pH-sensitive nanoparticules (NPs) that release the drug in the extracellular substance of the tumor or directly inside the malignant cells.1
The problems of chemotherapy Among the many problems of chemotherapy, there are three that we shall discuss here: (1) The bioavailability of the chemo drug at the tumor site; (2) An adequate penetration into the malignant cell; (3) Toxicity to normal cells.
The bioavailability of the chemo drug (CD) at the tumor site Once injected or ingested, the drug enters the circulatory system where it is distributed throughout the entire organism. For this, the drug must be hydrosoluble in order to be transported by the blood.2 For example, curcumin a very interesting nutraceutical for cancer treatment is not soluble in any kind of non-toxic solvents and thus, it cannot be used as a therapeutic substance. Once in the blood, the CD may be denaturalized by normal blood components or may be bound to proteins that decrease its activity. The goal of CDs is to achieve the highest possible concentration at the tumor site and the lowest possible concentration at normal tissues. If the drug can be “protected” from An Innovative Approach to Understanding and Treating Cancer: Targeting pH https://doi.org/10.1016/B978-0-12-819059-3.00022-8
© 2020 Elsevier Inc. All rights reserved.
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destruction (by macrophages) or inactivation (by other substances) during its widespread distribution throughout the organism, a higher concentration of active drug can be achieved at the required site of action. Nanoparticles (NPs) attached to CDs or CDs inside NPs can do this job. Another objective of treatment is the intracellular acidification of malignant cells with the goal of inducing acid stress and apoptosis, certain specialized NPs can do this too. But, how does this NP-CD complex know where to release the CD? This is the point where pH plays the capital role: NPs can be constructed in such a way that they release the drug specifically in sites with low pH, such as the extracellular space (ECS) of tumors. This are called the pH-sensitive NPs.
An adequate penetration into the malignant cell The NP “protects” the drug and releases it specifically at the tumor’s ECS. A second problem may ensue now: the acidic pH may inactivate the CD. Extracellular acidity has been found to be an important factor of chemoresistance. This is particularly evident in weakly basic anti-cancer drugs.3,4 Proton pump inhibitors5,6 and buffers like bicarbonate7 were proposed to modify this situation. But the relation between chemoresistance and pH is not limited to the extracellular acidity of tumors: the acidity of intracellular vesicles may play a role too. One of the mechanisms used by cancer cells to decrease the activity of chemotherapeutic drugs consists in sequestering them in endocytic vesicles.8 With certain CDs (e.g., doxorubicin) is not enough to carry them to the specific site of action, it is also necessary to avoid sequestration by protonation due to the acidic conditions of the tumor’s ECS. Using doxorubicin (DOX), Mellor and Calaghan found that P-gp (P-glycoprotein) and extracellular acidity represented similar and equivalent problems for an adequate anti-cancer effect. The effects of doxorubicin were limited to the outer layer of tumor cells and failed to reach the more hypoxic areas.9,10 Daunorubicin, doxorubicin and mitoxantrone are weak bases and therefore activity in a highly acidic medium is substantially reduced. Paclitaxel on the other hand is not affected by pH. Extracellular acidity is highly heterogeneous, with very acid areas coexisting with less acidic ones. This means that acid-sensitive chemotherapeutic drugs will achieve good results in less acidic areas, but will fail in the more acidic ones. The very acidic niches that remain unharmed by the treatment will become the core for relapse and/or clonal evolution toward resistance. Here again, pH-sensitive NPs can do the job by facilitating penetration into the cell and releasing the CD inside the cell. In this case, the design of the NP is different from the first situation.
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
Up to now, we have two different kinds of NPs: (a) NPs that release the CD in acidic sites, and (b) NPs that penetrate the malignant cell with its cargo releasing the drug inside the cell. There are also five other types of NPs in which we are interested: (c) an NP that can acidify the cytoplasm; (d) an NP that deposits its load to the cell surface but does not let it enter to the cell; (e) the double releasing NP; (f) the multidrug NP; (g) the multitarget NP (Fig. 1).
Fig. 1 Different types of NPs related to the pH gradient inversion: (1) pH-sensitive NP that releases the drug in acidic extracellular matrix; (2) pH-sensitive NP that facilitates drug penetration inside the cell; (3) pH-sensitive NP that acidifies the intracellular milieu; (4) pH-sensitive NP that deposits the drug on the cell surface but does not allow drug penetration inside the cell.
Toxicity to normal cells The above mentioned NPs are designed in such a way that drug release mainly takes place in the malignant tissues and by-passing the normal ones. And this is possible due to cancer’s altered pH homeostasis. In this chapter we will focus on these four types of pH-sensitive NPs: the ones that release the drug in acidic sites, the NPs that release the drug inside the malignant cell, the NPs that acidify the intracellular compartment and the NPs that concentrate the drug on the cell surface without allowing penetration. We will also touch briefly the other types of NPs.
pH-sensitive NPs that release the drug in acidic sites Many different nanocarriers have been tested in combination with chemotherapeutic drugs (CDs). These nanoparticles (NPs) are expected to increase the efficacy of
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therapeutic agents while reducing unwanted side effects. There are NPs that have been designed considering the advantage of the low pH of the extracellular space in tumors, being activated by this low pH and releasing the chemotherapeutic drug. This leads to a high accumulation of the drug at the tumor site.11 Hruby et al.12 developed a polymeric micellar system for the delivery of doxorubicin that released 43% of the drug at pH 5 and only 16% at pH 7.4. Meng et al.13 created a mesoporous silica NP for doxorubicin release at low pH. Many of these pH-activated delivery systems are engineered in such a way that they can reach lysosomes and nuclei, and be selectively released in the tumoral extracellular matrix. Pegylated liposomal doxorubicin is doxorubicin protected by a liposomal layer reducing the uptake by the reticulo-endothelial system and stably retaining the drug as a result of liposomal entrapment. These features increase its permanence in circulation, leading to an increased tumor uptake.14 It is not pH-activated, but it is a proof of concept that better vehicles for chemotherapeutic drugs can succeed in achieving improved results. In order to produce a maximal tumor concentration of a cationic drug such as doxorubicin (Dox) a specific nanocarrier must be engineered. Dox enters the cell through diffusion and interferes with the topoisomerase II-DNA complex leading to cell death.15 The idea of delivering NPs capable of transporting Dox unchanged at normal pH to sites were pH is lower like the tumor extracellular matrix, where the drug is released of this protection, is particularly attractive and it has been the object of great amount of research. The pH-activated drug release NPs are all based on the particular acid conditions of the pHe.16 Box 1 and Fig. 2.
BOX 1 pH-sensitive NPs must fulfill three criteria (1) Stability at physiologic pH. (2) Destabilization/degradation or conformational changes at pH <6.8. (3) Ability to release their load when pH falls below the trigger pH.
Fig. 3 shows the building of a pH-sensitive NP. Changes in pH introduce conformational changes in the NP that lead to disintegration or degradation of the NP thus releasing the drug.
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
Fig. 2 The pH-sensitive NP protects the drug while in the circulatory system. When it reaches an area with low pH, such as in tumors, it releases the drug. There is no drug release in normal tissues because their extracellular pH is markedly higher than in tumors. The small yellow circles in the blood vessel represent possible substances that can decrease the chemotherapeutic activity. The NP “protects” the drug against macrophages, blood proteins, etc.
Fig. 3 Schematic representation of a system of pH-activated drug delivery. The drug is “protected” during its journey through blood vessels and tissues with normal pH. It is released when it encounters an acidic pH.
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pH-sensitive NPs that release the drug inside the cell There are many mechanisms to drive the drug inside the cell. One of them consists in attaching a substance to the NP that has receptors on the cell surface. The substance binds the receptor and the internalization is carried out through the formation of an endosome. The endosomal pH is low and it releases the drug from the nanoparticle. The NP can be a polymer or a micelle charged with the drug, and there is a linker or cleavable bond that attaches the substance to the NP (Fig. 4).
Fig. 4 A mechanism to deliver a chemotherapeutic drug inside the cell. See explanation in the text.
For example, ovarian cancer cells over-express folate receptors in more than 90% of the cases.17 The inhibition of this folate receptors, essential for ovarian cancer metabolism, led to the development of Farletuzumab (a monoclonal antibody against folate receptor alpha). It failed in phase III clinical trials for relapsed ovarian cancer, but showed some minor benefits.18 An NP loaded with a chemotherapeutic drug such as a vinca alkaloid can be bound either to farletuzumab or to folate through a cleavable linker (Fig. 4, panel 1). This NP Vinca-Folate or Vinca-Farletuzumab targets the folate receptor at the cell membrane (Fig. 4, panels 2 and 3) and induces the internalization of the complex through the formation of an endosome (Fig. 4, panels 4 and 5). The acidic condition of the endosome (pH 5) breaks the cleavable linker and releases the drug (Fig. 4, panel 6).
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
Finally, endolysis (panel 7) releases the Vinca alkaloid in the cytoplasm where it acts against the microtubular system.19 Vintafolide or EC145 is the name of an experimental product fabricated on these parameters. Actually, this compound does not incorporate the vinca alkaloid into a micelle. It is directly attached to the pH-sensitive linker. It is in phase II Clinical Trial.20 In a phase I clinical trial Vintafolide showed low toxicity for the patients and very few side effects. Incorporating a micelle to the structure of Vintafolin, as seen in Fig. 4 would allow the internalization of more than one drug simultaneously. A step forward is the development of dual pH-activated nanoparticles, like the one proposed by Du et al.21 This dual pH system has the ability of reversing the surface charge of the particle when meeting the acidic tumor environment. In this manner the internalization of the particle is facilitated. When the particle inside the cell enters the endosomes, which are even more acidic than the extracellular environment, doxorubicin is further released. These dual systems protect the drug from the extracellular acidity of tumors, and facilitate its penetration inside the cell (Fig. 5).
Fig. 5 A dual pH-sensitive NP. (1) The NP-drug complex has a negative charge while circulating in the blood vessels. (2) When it leaves the blood vessels at the tumor site, the acidity of the tumoral extracellular matrix produces an electrical inversion that facilitates the penetration (3)–(5) into the cell. The extreme acidity of the endosomes further releases the drug (6) and (7).
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The intracellular delivery of substances by pH-activated nanoparticles is not exclusive for drugs, they can also be used to deliver other anti-cancer tools such as SiRNAs.22 Fig. 6
Fig. 6 A double coated NP delivery system for siRNA. (1) The first pH-sensitive coat drives the NP to the tumor’s ECS where it loses its first coat. The second coat is revealed with a strong negative electrostatic charge that increases the cellular uptake. (2) Inside the cell, the second coat is shedded and the siRNA is released.
shows a delivery system that transports siRNA instead of drugs. It has a double coating and loses the first coat when it arrives to a low pH region. At this point the negative charge of the second coat facilitates NP-siRNA’s entry to the cytoplasm. Then, the siRNA is released.
NPs that can acidify the cytoplasm It has been established that cytoplasmic acidification is the main objective for the reversal of pH alterations in cancer. Carbone et al.23 developed an NP in which they synthesized a chemical cage24 that shields a proton (H+) and coupled it to a gold nanoparticle. When this AuNP-caged H+ is taken up by the cell, stimulation with ultraviolet light releases the proton from the cagea and the freed H+ combines with CO3H producing carbonic acid that decreases intracellular pH (Fig. 7). a
“The idea behind the caging technique is that a molecule of interest can be rendered biologically inert (or caged) by chemical modification with a photoremovable protecting group”. From Ellis Davies.24
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
Fig. 7 (1) The NP consists of a chemically caged proton bound to a gold particle. (2) and (3) The NP is internalized into the cell. (4) Ultraviolet light stimulation “breaks” the cage releasing the proton that acidifies the cytoplasm.
These type of NPs have one drawback: their use would be limited to those tissues that can be irradiated with ultraviolet light. In the future, these NPs may become useful for treating oral cavity and oropharynx tumors. We also believe that it would be perfectly feasible to create endoscopes that can deliver UV light inside cavities.
NPs that deposit their load on the cell surface but do not let it enter the cell Inhibition of carbonic anhydrase (CA) is an important step in the reversal of the inverted pH gradient. The problem is that CA is a family of different isoforms. Those related to cancer are the membrane CAs: CAIX and CAXII. The model inhibitor of CAs is acetazolamide that works as a pan-inhibitor. This means that acetazolamide inhibits all the isoforms of CA, including cytoplasmic and membranous CAs. CAs are housekeeping enzymes and therefore fully inhibiting them all would be highly toxic. A drug delivery system able to inhibit only the membranous CAs of tumors without affecting normal cells would be the goal.25 There is advanced research with CA inhibitors attached to an NP that impedes cell penetration.26,27 These mechanisms would preserve intracellular CAs and deliver the drug mainly to malignant tissues that over-express membrane CAs. Fig. 8.
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Fig. 8 A non-permeable carrier transports the carbonic anhydrase inhibitor to the cell surface.
Double release NPs Fig. 5 is an example of a double release NP. When the NP reaches the tumor’s ECS undergoes a change in polarity that increases its penetration into the cell. Inside the endosome, the very low pH induces an NP’s conformational change with further release of the drug to the cytoplasm. This is just one example of many. Miao et al. developed a similar dual NP loaded with DOX.28 Kim et al.29 synthesized an interesting system of double targeting micelles transporting doxorubicin (DOX). In the first step these micelles protected DOX and facilitated its arrival to the cell surface. Then a second component of the micelle containing folate engaged the folate receptors of the cell membrane inducing endocytosis. The ensuing endocytolysis, released the drug. This system could be applied successfully to cells with the MDR phenotype. Actually this is a system of double targeting more than double release.
Multidrug release NPs NPs with the ability to deliver more than one drug have been developed recently.30 Liao et al.31 fabricated a system that delivers three chemotherapeutic drugs simultaneously:
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
doxorubicin, camptothecin and cisplatin. In this case the mechanism of release was not through pH-sensitive NPs but through light stimulation. In theory, a similar system could be developed with a pH-triggered release which would make the compound more focused on cancer cells.
Radiosensitizing NPs Ma et al.32 developed a NP based on the delivery of a carbonic anhydrase inhibitor and quercetin. This construction decreased the effects of hypoxia on tumors increasing the effects of radiotherapy. NP binding carbonic anhydrase is an interesting concept to deliver drug into hypoxic areas of the tumor.33
The making of an NP A useful pH-sensitive NP for oncological use must combine at least two basic features: (1) Ability to store the drug in stable conditions at normal pH. (2) Release the drug when the pH decreases below a pre-established threshold. These can be accomplished by adding chemical groups to the nanomaterial with the ability to: (a) accept or donate protons; (b) produce physical changes according to the pH. Polymer-based drug delivery Biopolymers (BPs) are the most frequently used nanocarriers for drug delivery. These compounds have some features that make them the best choice in the case of pHsensitive systems: (1) BPs are biodegradable. (2) BPs are compatible with human tissues and body fluids. (3) BPs are easy to manipulate in the laboratory.34 (4) BPs have the ability to respond to ionization with conformational changes. Most of the pH-sensitive NPs are actually pH-sensitive polymers that can respond with changes in their physical properties when they encounter changes in the pH of their environment. In many of these NPs the polymer is dissolved when subjected to certain pH changes.35 This is the case of NPs made of poly(β-amino) ester shown in Fig. 9.36 These polymers can transport the drug in two different ways37: (1) the drug is physically entrapped in the NP; (2) The drug is covalently bound to the NP (Fig. 10). In the second case the drug is bound to the surface of a nanocarrier through a covalent acid-labile bond. The chemical bond is hydrolyzed when the pH decreases, releasing the drug.
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Fig. 9 Degradation or disassembly of the NP releases the drug when pH is lowered from the physiologic to the acidic range.
Micelle-based drug delivery Another example of disassembly induced by low pH is the fabrication of NPs formed by micelles that are bound to hydrophobic drugs by a linker. The acidic pH dissolves the micelles releasing the drug (Fig. 10).
Fig. 10 Upper panel: two different mechanisms of drug transport by a polymer: inside the nanocarriers (left) or attached through a chemically labile bond (the linker) (right) Lower panel: a pH-sensitive micellar NP.
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
Polyester based NPs release acidic degradation products. This may represent a drawback if the degradation products remain in the ECS increasing its acidity. Hefferman et al.38 developed an NP based on a new hydrophobic polymer called polyketal that avoids the problem. The possibilities for the development of new site-specific pH-sensitive NPs for the delivery of drugs and other types of molecules are enormous and we are just at the beginning. Table 1 is only a small sample of pH-sensitive NPs.
Table 1 A summary of pH activated drug delivery systems for cancer treatment Reference
Yang et al. (2018)
Mechanism 39
Ju et al. (2017)40
Ehi-Eromesele et al. (2017)41 Guo et al. (2017)42 Wu et al. (2017)43
Li et al. (2016)44
Jiang et al. (2017)45
Piao et al. (2017)46 Gisbert-Garzaran et al. (2017)47 Zignani et al. (2000)48
Poly-L-lysine-lipoic acid modified by dimethylmaleic anhydride used as a carrier for doxorubicin with enhanced cell internalization and intracellular pH release. A liposome prepared with a synthetic oligopeptide lipid, soy phosphatidylcholine, and cholesterol showed that this pH sensitive drug carrier enhanced tumor cell uptake and increase cytoplasmic distribution. Magnetic nanoparticles coated with silica, pH sensitive particles were synthesized as carriers of 5 FU. At pH 5 this NP released three times more 5 FU than at pH 7.4. A gene transfection system was engineered in which the gene is part of a pH-responsive charge-convertible ternary complex. A graft copolymer of poly(butylenes succinate)-g-cysteamine-bi-poly (ethylene glycol) was synthesized for doxorubicin transport. This micellar system could switch from less than 25% to 84% of doxorubicin release according to the media pH. A dual sensitive biodegradable micelle for drug transport was built with maximal release of the drug inside the tumor cell. The micelle was sensitive to pH and redox conditions. The micelles were quickly and fully internalized by cellular endosomes. A transactivator of transcription (TAT) nanoparticle was developed with increased intracellular retention and nuclear translocation of a platinum based chemotherapeutic drug. The intranuclear acid conditions triggered the TAT activity (not the extracellular acidity). Gold nanocages with zwitterionic pH sensitive coating were used to increase delivery of chemotherapeutic drugs to the tumor. pH-responsive mesoporous of silica and carbon was used to increase delivery of chemotherapeutic drugs into tumors. pH-sensitive release of drug was achieved with egg phosphatidylcholine liposomes adding N-isopropylacrylamide (NIPA) copolymers.
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Clinical implications Chemotherapeutic drugs are generally very toxic. Chemotherapy can be defined as a “controlled intoxication” where art and science join to keep the drug within a narrow therapeutic range. If that range is exceeded the patient will suffer serious adverse effects and if the range is not achieved the treatment will be ineffective. Therefore, the way of administering the drug (drug delivery system) is of capital importance.49 The other main concern in oncology is to deliver the highest possible amount of drug to the tumor and ideally none to the rest of the organism. Nano-systems that are pH-dependent will be a part of the new delivery systems that may eventually change oncological practice as we know it today. Liu et al.1 wrote: “Stimulus-responsive nanomaterials are termed as ‘smart’, ‘intelligent’, or ‘environmentally sensitive’, and have greater potential than traditional delivery systems.” A few interesting objectives can be achieved by using pH-sensitive nanocarriers: (1) Specific and focalized delivery of drugs to the tumor without toxicity to normal cells. (2) Intracellular acidification limited to cancer cells. (3) Counteracting or by-passing multidrug resistance (MDR). (4) Intracellular delivery of siRNAs that can block oncogenic pathways and proteins. (5) Increased stability and permanency of the drug in circulation. (6) Higher concentrations of the drug without generating intolerable toxicity. (7) Increased retention of the drug inside the tumor. These compounds are not yet in clinical use, but they will be soon. The hand-foot syndrome (palmar-plantar erythrodysesthesia) is a well-known adverse event with some cytotoxic CDs50,51 including the non pH-sensitive pegylated liposomal doxorubicin (PLD). The incidence with PLD is close to 50%.52 This problem results in treatment interruption in many cases. However, it is less frequent when DOX is used alone.53 This led to the conclusion that prolonged circulation due to the pegylated liposome NP increased leakage to non tumor-specific sites.54 As a speculation, we may assume that using pH-sensitive NPs instead of PLD, the leakage probably would not be avoided. However the leaked NPs would not release DOX and therefore a foot and hand syndrome would be prevented. The advantages of a pH-sensitive NP chemotherapy drug delivery become very evident with the example described above.
Conclusions We have oversimplified this chapter, sacrificing scientific details for the sake of clarity to offer a better understanding of this complex topic. The first nanoparticles (NPs) were mainly designed for drug delivery improving its pharmacokinetics, prolonging its presence in the circulatory system and decreasing its
Taking advantage of cancer’s pH alterations: pH-sensitive nanoparticles
degradation or inactivation before reaching its target. Then, new and more ambitious objectives were set for NPs: more focused delivery to the tumor, multidrug delivery, siRNA delivery, specific intracellular targeting, controlled drug release, environmental sensitive response, etc. After the success of the pegylated liposomal doxorubicin (PLD), that has been used intensively in the last 10 years, a considerable effort has been dedicated to pH-sensitive drug carriers and very interesting developments have been achieved. The pH paradigm of cancer is a targetable soft spot of cancer that exposes malignant cells to an easier attack by pH-sensitive nanocarriers. However, none of these new pH-sensitive carrier systems have yet been approved for human use. It is highly possible that we shall see breakthroughs on this issue in the near future. It opens the possibility to target certain components of the pH paradigm that, at present, lack efficient blockers. This would be the case of NBC (sodium/bicarbonate cotransporter), just to mention one example. As a membrane protein we think it may be targeted with nanocarriers that can protect normal cells from the attack. The many broad and interesting possibilities that nanocarriers can offer to the world of oncology have not yet been fully explored. We trust that these tools will become part of clinical oncology very soon. This chapter is only a brief glance at this subject that would require—and deserves—many dedicated volumes. Peer et al.55 wrote: “Nanotechnology has the potential to revolutionize cancer diagnosis and therapy. Advances in protein engineering and materials science have contributed to novel nanoscale targeting approaches that may bring new hope to cancer patients.” We share this concept.
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