PEG-I-dC₁₆ Acid-sensitive Copolymeric Micelles For The Targeting Drug Delivery System

pH-sensitive amphiphilic poly （ethylene glycol）–imine–benzoic–dipalmitate (PEG-I-dC₁₆) polymers with two different PEGs molecularweight (PEG_2k,PEG_5k) were designed and synthesized. Poly （ethyleneglycol）-amide-benzoic-dipalmitate (PEG-A-dC₁₆) polymers weresynthesized as a comparison. The molecular structures of the polymerswere confirmed with1H NMR. The micelle formation of block polymersin aqueous solution is commonly demonstrated by fluorescence probe.CMC values of PEG_2k-I-dC₁₆, PEG_2k-A-dC₁₆, PEG_5k-I-dC₁₆andPEG_5k-A-dC₁₆polymers were0.006mg/mL,0.007mg/mL,0.023mg/mLand0.036mg/mL, respectively. Compared to the values, CMC valuesdecrease with the less hydrophilic PEG segments.These block polymersreadily self-assembling into core/shell micelles in water with averagesizes of about35-50nm were determined by dynamic light scattering（DLS）. Doxorubicin （DOX） was loaded into the core of these micelleswith a high drug loading capacity. DOX was encapsulated in the micellesusing membrane dialysis method. Using10wt%to30wt%of DOXrelative to polymer, all polymer micelles showed good loading capacitiesfor DOX and LC values were in the range of7.0wt%to13.1wt%were typically obtained.The PEG-I-dC₁₆micelles exhibited some pH-sensitive characteristic.The in vitro release showed an unusual pH dependent release behavior,that is, significantly faster drug release at mildly acidic pH of6.5compared to physiological pH, at the pH5.0, the rate of DOX releasewere much slower than that of at both pH7.4and pH6.5and redprecipitate was observed in the dialysis bag. Interestingly, under the samerelease conditions, no red precipitate was found at pH5.0in the dialysisbag for PEG-A-dC₁₆micelles.We predict the possible reason may formcomplex between drug and the polymeric residues of removing PEGs inthe media, which could alter the DOX’s solubility in the release mediumand hinder the permeability of free DOX out the dialysis.Cytotoxic effects of the polymers, free DOX, or DOX-loadedmicelles for HepG2and A549cells were measured by MTT assay.Toxicity test showed that the polymers had no significant inhibitoryeffects on A549cells, even at high concentration （4mg/mL） whereas theDOX-loaded micelles remained high cytotoxicity for HepG2and A549cells. the probably reason was that free DOX can be readily transportedand diffuse into cytoplasm by passive diffusion, while drug loadedmicelles have to be internalized by endocytosis and then release the druginto cytoplasm, which usually delay the nuclear drug delivery.The cell uptake of PEG-I-dC₁₆micelles was measured by Flow cytometry. The results showed that the mean fluorescence intensityincreased in the cells incubated with PEG-I-dC₁₆/DOX micelles from0.5h to2h. The acid liable linker of PEG-I-dC₁₆micelle may enhance thedrug release in endsome or lysosome and increase the cellular uptake ofDOX in the process from endocytosis to exocytosis process. The cellularuptake extent of PEG_2k-I-dC₁₆was greater than that of PEG_2k-A-dC₁₆.PEG-I-dC₁₆micelles showed more efficiently deliver drug intonucleus of HepG2cells by confocal laser scanning microscopy than thatof PEG-A-dC₁₆micelles. PEG_2k-I-dC₁₆/DOX appeared more drugaccumulation in nucleus at0.5h than that of free DOX, whilePEG_2k-A-dC₁₆/DOX appeared hardly any drug in the nucleus. At2h,DOX was released from PEG_2k-I-dC₁₆micelles and almost concentratedin the nucleus of HepG2cells, PEG_2k-A-dC₁₆encapsulating DOX hardlyexpose to cell nucleus for given time, indicating relative fewer DOX wasreleased from the PEG_2k-A-dC₁₆/DOX micelles without acid labilelinkage. This observation might be attributed to the PEG-I-dC₁₆/DOXmicelles through endocytic pathways. All these characteristicsdemonstrated that this system might have potential applications inanticancer drug delivery. Drug escaping from lysosome and the antitumoractivity in tumor grafted mice in vivo will be further investigated.