high metabolic rate but inadequate removal of lactic acid [128–134] and carbon dioxide [135], as well as other mechanisms [136]. Electrical and chemical probes showed that the mean pH of tumor extracellular fluid is typically about 7.06 with a range of 5.7–7.8 [137]. Low pHe benefits tumor cells because it increases drug resistance by slowing the uptake of weakly basic drugs such as DOX and reducing their effects on tumors, promotes invasiveness [138], and induces vascular endothe- lial growth factor (VEGF) [139]. On the other hand, this acidic pHe can be exploited as a drug-release trigger.
For example, Bae and coworkers reported pH-induced destabilizable poly (l-histidine) (PHis)-based micelles targeting the tumor acidic environment [140].
PHis has a pH-dependent water-solubility as a result of protonation of the unsatu- rated nitrogen of its imidazole ring at low pH [141]. Its soluble/insoluble transition pH (pHt) is dependent on its molecular weight. PHis with a Mn more than 10 kDa becomes soluble at pH lower than 6 [141], but PHis with a Mn of 5 kDa becomes soluble at pH of about 6.5 [140]. Introducing water-soluble groups or polymers increases the pHt [140, 142]. For instance, PHis5k-block-PEG2K (PHis-PEG) had a pHt of about 7.0 [140]. This PHis-PEG formed micelles with diameters of about 100 nm. The critical micelle concentration (CMC) was dependent on the solution pH (Fig. 10.3). The micelles had low stability at pH lower than 7 due to the protona- tion of the hydrophobic PHis core (Fig. 10.4). When the solution pH was less than 5, no micelles could be detected.
As a result of this pH-dependent stability, the PHis-PEG micelles loaded with DOX exhibited a pH-dependent drug release. DOX was released faster at pH lower than 7.0 than at pH 7.4 and 8.0 (Fig. 10.5) [143]. It was hypothesized that PHis- PEG micelles were destabilized in a slightly acidic environment and hence released the DOX into the extracellular medium for enhanced drug permeation into the tumor cells due to high concentration gradients. The in vitro cytotoxicity was tested
5.0 0 50 100
CMC (µg/mL)
pH 150
5.5 6.0 6.5 7.0 7.5 8.0
Fig. 10.3 The pH effect on the CMC of PHis5K–PEG2K micelles. (reproduced from [140], with permission from Elsevier)
by incubating the DOX-loaded micelles with A2780 cells at pH 7.4 and 8.0. When cells were cultured with DOX/PHis-PEG at pH 6.8, a higher fraction of cells took up DOX, with a higher intracellular DOX concentration, and correspondingly lower cell viability, than when cultured at pH 7.4, as shown in Fig. 10.6 [144], which suggested that more DOX was available at the lower pH [144]. Also, compared with free DOX, the DOX-loaded micelles had a longer retention of DOX in blood, a higher DOX concentration in the cancer tissue, and more pronounced tumor
96
94
92 98 100
Transmittance %
pH 102
6.0 6.5 7.0 7.5 8.0
Fig. 10.4 The transmittance change of PHis5K-PEG2K micelles (0.1 g/l) with pH. (reproduced from [140], with permission from Elsevier)
100
Cumulative DOX Release (%)
Time (hr) 80
60
40
20
0
0 10 20 30 40 50
Fig. 10.5 pH-dependent cumulative DOX release from PHis-PEG micelles at pH 8.0 ( ), 7.4 (), 7.2 ( ), 7.0 ( ), 6.8 (), 6.2 ( ), and 5.0 ( ). (revised and reproduced from [143], with permission from Elsevier)
suppression. It was presumed that the DOX release triggered by tumor extracellular pH (pHe, 7.0) from the DOX/PHis-PEG accumulated in the tumor via the EPR effect contributed to the tumor inhibition.
As the data from Fig. 10.5 suggests, the PHis-PEG micelles are unstable and can release their contents at neutral pH. In order to deal with this problem, an amphiphilic block copolymer, PLLA-PEG, was added to make mixed micelles, in which the core was expected to contain poly(l-lactic acid) (PLLA) and PHis chains. The PLLA block in the core stabilized the micelles and hence suppressed the drug release at the near neutral pH. The optimum content of PLLA-PEG was found to be about 25–40% (Fig. 10.7). The DOX delivered from such mixed micelles showed low cytotoxicity at pH above 7.0 but high cytotoxicity at pH ∼ 6.8 [143].
0.0001
Free DOX pH 6.8 pH 7.4
Cell viability (%)
DOX (mg/ml) 0
20 40 60 80 100
0.001 0.01 0.1 1
Fig. 10.6 Cytotoxicity of free DOX and DOX/PHis-PEG micelles at pH 7.4 and 6.8 after a 48-h incubation. (reproduced from [144], with permission from Taylor and Francis Ltd)
5.0 20 40 60 80 100
5.5 6.0 6.5 pH
Total ADR Release (%) in 24 hrs 7.0 7.5 8.0
Fig. 10.7 pH-dependent 24-h-cumulative DOX release from the mixed micelles composed of PHis-PEG and PLLA-PEG with wt.% of PLLA-PEG at 0 ( ), 10 (), 25 ( ), and 40 ( ). (reproduced from [143], with permission from Elsevier)
Another approach to tuning the pH-sensitivity proposed by Bae and coworkers is to introduce sulfadimethoxine (SDM; pKa = 6.1; Scheme 10.1a) as a pH-sensitive motif [145, 146]. Sulfonamide is a weak acid because the proton of sulfonamide can be readily ionized to liberate a proton in basic solution [145, 146]. Thus, dif- ferent from the amine-based PHis, SDM becomes hydrophobic by protonation at low pH. For example, pullulan acetate (PA) (Scheme 10.1b) conjugated with SDM moieties (PA/SDM) formed hydrogel nanoparticles that showed good stability at pH 7.4, but shrank and aggregated below pH 7.0, at which pH SDM became hydro- phobic. As a result, the DOX release rate from the PA/SDM nanoparticles was low around the physiological pH but significantly enhanced below pH 6.8 (Fig. 10.8), which was consistent with the core structure changes observed using a fluorescent probe and a microviscosity probe [147].
The cytotoxicity of the DOX-loaded PA/SDM nanoparticles was evaluated using a breast-tumor cell line (MCF-7) to test the feasibility of the nanoparticles in target- ing acidic tumor extracellular pH [148]. DOX-PA/SDM nanoparticles at pH 6.8 showed cytotoxicity similar to free DOX but higher cytotoxicity than the particles at pH 7.4. This pronounced cytotoxicity of the nanoparticles at low pH was partially attributed to the accelerated release of DOX triggered by pH changes. Another contributing factor, on the basis of the confocal laser microscopy characterization, was that at pH 6.8 and 6.4, SDM deionization caused the nanoparticle surfaces to become hydrophobic and thus aggressively bind to the cell membranes [148]. The cancer targeting and cellular uptake were promoted by introducing targeting groups such as vitamin H (biotin) [149].
Although the drug release from such pH-responsive nanoparticles triggered by the cancer pHe can substantially reduce the systemic toxicity and enhance in vitro and in vivo anticancer activity, it does not solve the drug resistance problem. This is because cancer cells can lose the drug receptors to slow down drug uptake and
H2N S NH
N N
OCH3
OCH3 O
O OH O
OH O H3CCO
CH2
O O
OH OH O CH2OCCH3
O
(a)
(b)
O OH O
OH O H3CCO
CH2
O O
OH OH O CH2OCCH3
O O
OH OH O CH2OCCH3 O
OH OHO O CH2OCCH3
n
Scheme 10.1 The chemical structures of (a) sulfadimethoxine (SDM) and (b) pullulan acetate (PA). (modified from [146], with permission from Springer)
overexpress ATP Binding Cassette (ABC) transporters in their plasma membranes to efficiently transport cancer drugs out of the cells as they are entering the cell membranes [6, 10, 12, 13, 26–28], and thus reduce the cellular drug accumulation [29]. Therefore, the drug released in the extracellular fluid is difficult to reach the cytoplasm.