Micelles, Dox-loaded

Dox-Loaded Micelle with Intracellular pH-Triggered Drug Action. 123... 

The concentration of DOX in plasma after injection of free DOX in animal models ranges from several micrograms per milliliter to 20 pg/mL after 5-10 min at a dose of 10 mg/kg [81-83]. One study reported a DOX half-life of 26 min [81]. As compared with the values of free DOX, DOX that was carried by PEG-po-ly[Asp(ADR)] micelles demonstrated a high initial concentration in the blood (and plasma), with a long half-life of 70 min. This result indicates that the PEG chains in the outer shell play a crucial role in the extended circulation of PEG-P[Asp(ADR)] micelles in the blood and promote less absorption of DOX-loaded micelles into living tissues and less metabolic activity by the tissues than free DOX [84]. 

In another study, an amphiphilic hyperbranched multiarm copolymer [H40-star-(PLA-b-PEP-OH)J was synthesized through a two-step ROP procedirre (Figure 12). First, Boltom H40 was used as the macroinitiator for the ROP of l-1A to form the intermediate (H40-star-PLA-OH). Then, the ROP of EEP was further initiated to produce H40-star-(PLA-b-PEP-OH). Benefiting from the amphiphilic stmcture, H40-star-(PLA-b-PEP-OH) was able to self-assemble into micelles in water with an average diameter of 130 nm. In vitro evaluation of these micelles demonstrated their excellent biocompatibility and eftident cellular uptake. DOX-loaded micelles were investigated for the proliferation inhibition of HeLa human cervical cardnoma cell line, and the DOX dose requited for 50% cellular growth inhibition was found to be 1 pg ml". ... 

For the DOX-loaded micelles, DOX was first neutralized before micelle preparation. 3.0 mg of DOX was neutralized with an excess mount of TEA in 1.0 mL of THF. The DOX solution was then added into the 2.0-mL THF solution of ABA copolymer (20 mg). This solution was added 1.5 mL of double distilled water under stirring for 6 h. To remove untrapped DOX and TEA, the mixture was next transferred for dialysis against double distilled water for 24 h to produce micelles. The water was replaced hourly for the first 3 h. 

The size and the size distribution of copolymeric micelles were measured with dynamic light scattering, and the results are demonstrated in Fig. 1. The effective diameter of the unloaded-micelles was 73.5 nm with narrow size distribution (polydispersity = 0.053). The size of the DOX-loaded micelles increased to 123.2 nm compared with 73.5 nm of unloaded-micelles. It was recognized that the particle size of DOX-loaded micelles increased slightly comparing to that of the DOX-free micelles. Similar results were also obtained in other report [5],... 

The in vitro release profiles of DOX from the polymeric micelles was studied in in PBS (0.1 M, pH 7.4) and acetate buffer solutions (0.1 M, pH 5.4) at 37 °C. The results showed an initial burst release of DOX and followed by a sustained release for about 48 h. The initial burst release of DOX from micelles could be attributed to the diffusion of DOX located close to the surface of particles or within the hydrophilic shell [6]. The total release of DOX in a period of 48 h with pH 7.4 and 5.4 was 25% and 37% of total DOX concentration, respectively. However, the release of DOX at a pH value of 5.4 was found faster than that at a pH value of 7.4. These results could be attributed to the re-protonation of the amino group of DOX and the faster degradation of the micelle core at lower pH values. This pH-dependent release profile is of particular interest. It is expected that the greater part of DOX-loaded micelles will remain in the micelles cores for a considerable time period in plasma after intravenous administration and have the potential for prolonged DOX retention time in the blood circulatioa However, a faster release may occur at low local pH surrounding the tumor site or by the more acidic environment inside the endosome and lysosome of tumor cells after cellular uptake of micelles through endocytosis. 

The oil-in-water emulsion method consists Lrst of preparing an aqueous solution of the copolymer. To this a solution of the drug in a water-insoluble volatile solvent (e.g., chloroform is added to form an oil-in-water emulsion) (Jones and Leroux, 1999). The micelle-drug complex forms as the solvent evaporates. The main advantage of the dialysis procedure over this method is that potentially toxic solvents can be avoided. Both dialysis and oil-in-water emulsion methods were compared for the incorporation of DOX in PECb-PBLA micelles (Kwon et al., 1997). The emulsiLcation method was more efLcient with a DOX loading of 12% (w/w) (Kwon et al., 1997) compared with 8% (w/w) for the dialysis technique (Kwon etal., 1995). 

Kwon et al. (1997) applied reversed-phase HPLC to determine doxorubicin (DOX) loading in PEO-bPBLA micelles. Samples of 20 mL diluted to jk /mL DOX with 0.10 M sodium phosphate buffer, pH 7.4, were separated af4Dat a low rate of 1.0 mL/min. The mobile phase was a linear gradient mixture of an aqueous solution of 1 % acetic acid and ACN (15% v/vto 85% v/v). Detection of DOX was done by measuring its UV absorbance at 485 nm. 

In particular, the system was able to overcome drug resistance and cross the blood-brain barrier. Preclinical studies on the DOX-loaded pluronic micelles SP1049C revealed superior tumor inhibition and extended lifespan to free drug. Phase I clinical studies showed a lower degree of DOX toxicity for SP1049C than for the free DOX, and three of 21 patients treated with SP1049C had transient partial responses. ... 

Bae and coworkers used PHis-PEG (75wt.%)/PLLA-PEG-folate (25wt.%) to make pH-sensitive micelles with folate-targeting groups (PHSM/f). The in vitro and in vivo anticancer activities of DOX-loaded PHSM/f were evaluated using... 

MCF-7 cells and their xenograph tumors, and compared with DOX-loaded pH-insensitive micelles made of PLLA-PEG with folate targeting groups (PHlM/f) [99]. The cellular localization of the nanoparticles was confirmed by confocal microscopy (Fig. 10.13). DOX delivered by PHSM/f was found uniformly distributed in the cytosol as well as in the nucleus, while DOX/PHlM/f was entrapped in endosome and multivesicular bodies. It was thus hypothesized that PHis, which is known to have an endosomal membrane-disruption activity induced by a proton sponge mechanism of its imidazole groups [209, 210], disrupted the compartment membrane and released DOX into the cytosol. As a result, DOX/PHSM/f showed much higher in vitro and in vivo anticancer activities toward DOX-resistant cells (Fig. 10.14). 

Fig. 2 A first generation of drug-loading micelles, a Schematic illustration of the formation of polymeric micelle of Dox-conjugated PEG-PAsp block copolymer. Additional Dox can be physically entrapped in the micelle, b Chemical structures of PEG-PAsp block copolymer and Dox...

Fig. 4 A second generation of the drug loading micelle with a pH-sensitive drug releasing property, a Formation of pH-sensitive polymeric micelles from PEG-(PAsp-Hyd-Dox) block copolymers. Antitumor drugs (Dox), conjugated through acid-labile hydrazone linkers, are released in lower pH conditions, b Time- and pH-dependent Dox release profile from the micelles. The micelles selectively release Dox under the pH condition of region B, which corresponds to the intracellular environment. The amount of loaded Dox in the micelles was calculated from the released Dox at pH 3.0 where all of the loaded drugs were assumed to be released from the micelle...

Acid-cleavable DOX-loaded biodegradable polymeric micelles were also made by copolymerization of BMDO and l,2 3,4-di-O-isopropylidene-6-O-(20-formyl-40-vmylphenyl)-D-galactopyranose (IVDG) by controlled radical (RAFT) polymerization. The anticancer drug DOX was conjugated to the deprotected copolymer (after removal of the isopropylidene group) via an acid-labile Schiff base linkage [68]. 

Figure 16 Structure of MPEG-HPAE block copolymer. DOX-loaded MPEG-HPAE micelles under physiological pH were completely dissociated and rapidly released DOX in weakly acidic pH environments. (Reproduced from Ref. 73b. Elsevier, 2007.)...

In addition, there are reports suggesting that, through the application of ultrasound, the degradation times of biodegradable polymers can be increased (Kost et al., 1988). As an example, pluronic block copolymer micelles have been tested in combination with ultrasound, and an increased drug release rate was observed (Munshi et al., 1997). Interestingly, ultrasound has also been shown to increase cellular uptake and nucleus internalization of DOX-loaded pluronic micelles (Marin et al., 2001). 

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