Stepwise PH-targeting Oral Nano-carriers:Preparation,Characterization In Vitro And In Vivo

Chemotherapeutics have been widely useful in clinical treatment of cancer,and relevant studies have shown that they can inhibit the growth of tumor cells and induce apoptosis.However,the poor solubility and absorptivity as well as complex physiological barrier will seriously affect the tumor therapeutic effect of drugs in vivo.Therefore,it is necessary to use drug delivery for sending loaded-drug into the body,and then release this anti-cancer agent under a certain environment to avoid the systemic toxicity caused by chemotherapy.The researchers design a series of drug delivery for oral or intravenous treatment that could work well,for example,oral drug carriers for targeted absorption by stomach or intestines;nano-drug delivery carriers(nano-DDS)for intravenous treatment.Both of these two methods of administration have corresponding advantages:oral administration is convenient and simple,as well as the biological safety is high;intravenous administration can achieve the maximum bioavailability,and this delivery plays a faster role.However,there are some unavoidable drawbacks in drug carriers that are modified only for oral or intravenous delivery alone:oral administration has a strong first-pass effect that leads to lower bioavailability and higher drug toxicity;intravenous administration causes a very low biosecurity because it goes directly through the skin into blood with massive chemotherapeutics,and the body may not be able to withstand rapid changes in drug concentrations.Besides,there is an obvious pH-gradient due to between the oral delivery and the intravenous injection system.Hence,the advantages of intravenous and oral administration can be integrated by reasonably designed stepwise pH-targeting carrier to avoid that of disadvantages.In this paper,we intend to prepare an oral nano-scale drug carrier with double-layer structure.The out-layer is a carboxymethyl chitosan(CMC)coating,which can protect the inner drug-carrying core from degradation in the strong acid environment of stomach for protecting gastrointestinal tract to maximum extent.CMC-coating then targeted small intestinal epithelial cells and open tight junctions(TJs)to facilitate absorption.The inner part is the hydrophobic core consist of poly(ortho ester urethane)(POEU)and anti-cancer agent,which can effectively make the nanoparticles steadily circulate for a long time after this delivery is absorbed into the blood.This nanoparticle can be effectively triggered tumor tisse by enhanced permeability and retention(EPR)effect of the blood vessels of cancer tumor,and then,nanoparticle was disintegrated due to the cleavage of ortho ester under the weak-acid environment(pH 5.0).Which ultimately leads to the rapid release of encapsulated drug in cancer cells to obtain considerable cell lethality through minimal systemic toxicity.In this experiment,POEU was successfully synthesized by two-step polymerization under mild conditions,which was equally consist of acid-sensitive dihydroxyl ortho ester monomer and hydrophobic polycaprolactone(PCL,Fw=530).It was characterized by nuclear magnetic resonance(NMR)and gel permeation chromatography(GPC),relative results shown that this polymer was correctly synthesized and the molecular weight(Mn=1.81×104)was moderate.And then,different nanoparticles were fabricated by commonly used oil in water ultrasonic emulsification technology:the single layer nanoparticles(designed as NP1)prepared by polyvinyl alcohol(PVA),besides,CMC as the emulsifier for preparation of double-layer nanoparticles(designed as NP2).The different morphology of NP1 and NP2 can be observed by transmission electron microscopy(TEM)and scanning electron microscope(SEM),and the study found that NP2 is a double layer structure.The dynamic light diffraction(DLS)test shows that the hydrated particles of NP1 and NP2 are 196.3 nm and 359.8 nm,respectively.Fourier Transform Infrared Spectrometer(FT-IR)and NMR were used to characterize both NP1 and NP2 and observe corresponding ion buffer capacity,these results indicate that NP2 is a mixture of NP1 and CMC,and CMC is located outside.Then the stability of nanoparticles was verified by different simulated physiological environments and in vivo experiments.When NP1 and NP2 were incubated with in vitro strong acid environment(pH 1.0)and simulated gastric fluid(SGF),a completely different performance appeared,NP1 was quickly disintegrated leads to the suspension precipitate,which can be attributed to rapid fracture ortho ester bonds,while NP2 suspension was stable with strong acid tolerate.For further investigated the in vivo stability of NP2,we can observe a stable nanoparticles structure in mice serum after a high dose of NP2 was orally seed into mice,indicated that NP2 could be absorbed by intestinal epithelial cells as drug carrier without obvious degradation phenomenon.Of course,as a drug carrier,excellent biocompatibility and biosafety are essential,so the toxicity of the drug carrier needs to be evaluated in vitro and in vivo.NIH/3T3 cells MTT experiments in vitro showed that neither NP1 nor NP2 could cause any inhibitory effect on the growth of plane cell,and the cell viability of cells incubated with different concentrations can reach more than 95%.Meanwhile,the acute toxicity of mice was studied by different doses of nanoparticles,these results shown that even after super-high dose(2000 mg/kg)of NP1 or NP2 treatment,the mice was normal growing and relative section of heart,liver,spleen,lung,kidney,intestine and stomach showed that negligible-toxicity was occurred at each organ.Furthermore,the plasma biochemical indexes of each groups were abnormal after treated for 14 days.Oral doxorubicin(DOX)has the greatest side effects on the heart.It is necessary to improve this phenomenon as much as possible,so we need to prepare a kind of oral dosage form of nano-drug-carrier,and they are named as NP1/DOX and NP2/DOX,respectively.It was found that the drug loading rate(DLC)of NP1/dox and NP2/dox was 14.32%and 17.27%,respectively,indicated that NPs/DOX could be formed at a higher rate of drug loading.Besides,drug release studies shown that both NP1/DOX and NP2/DOX were able to deliver drugs in a stable and controlled manner under low-pH value(5.0),which ensure that drug released from NPs/DOX could be trigger by weak-acid in cancer cells.It is more important that oral DOX-delivery should possess a considerably high oral-bioavailability and rational drug distribution in vivo,so,in this experiment,the oral-bioavailability of free DOX,NP1/DOX and NP2/DOX was studied.In addition,the bioavailability of intravenous free DOX at 6 mg/kg was set as 100%.We found that the bioavailability after treat by NP2/DOX was 75.4%,which was 3.93-folds and 2.77-folds of the oral bioavailability treated by free DOX and NP1/DOX,respectively,indicating that this double-layers drug-loaded nanoparticles can significantly improve the oral doxorubicin bioavailability.In the same way,the drug distribution in main organs of three oral treatment groups was measured after different administration time,it was found that the drug content in the heart was higher after oral administration of free DOX and NP1/DOX.Both the maximum value exceeded 4 ?g/g tissue,and there was a high risk.On the contrary,the DOX concentration level in tumor tissue are low.In the NP2/DOX group,however,corresponding results were different.More concretely,drug concentration of heart was no more than 1.6 ?g/g tissue during whole treat period,and the DOX concentration in tumor tissue was the highest that other different dosage,and the maximum value close to 8 ?g/g tissue,which contributed to NP2/DOX can enter the blood vessels as drug delivery by absorption of small intestinal epithelial cells,and then they were enriched into tumor tissue by EPR effect.Last but not least,the actual performance in vivo of NPs/DOX was assessed by analyzed of the inhibition of solid tumors and the toxicity effects on main organs during whole treatment.Briefly,we set up a mouse model of liver cancer,then free DOX,NP1/DOX and NP2/DOX at a dose of 10 mg/kg was used for continuous oral treatment every single day.In addition,saline,empty NP1 and NP2 was selected as negative control group,and it was found that there were no significant different in every negative control group even after continuous gastric treatment for a week,which further verified the excellent physiological compatibility of these delivery.For free DOX and NP1/DOX treated groups,although there is a certain inhibition on tumor tissue compared to negative control groups,it is not obvious.Additionally,relative cardiac and gastrointestinal sections shown that these DOX formulation can cause extreme systemic toxicity.Besides,the tumor volume and weight of NP2/DOX treatment group decreased significantly,which displayed different symptoms compared with other treatment groups.The weight was only 0.28 g after continuous treatment for 7 days,which was 14.99%,19.60%and 19.95%of the tumor weight after saline,free DOX and NP1/DOX treatment,respectively.And the paraffin section exhibited that this area of cell damage in tumor tissue was largest and the most obvious.Most importantly,stomach and intestine sections of this group showed no significant damage after treatment on an account of the super-stability of NP2/DOX in gastrointestinal tract.Corresponding to the heart section in control group,the morphology of NP2/DOX treated groups is similar,indicating that this double-layers drug-delivery did not produce anymore cardiotoxicity.To sum up,this double-layers DOX-loaded nanoparticles(NP2/DOX)can reach super-high bioavailability(75.4%)similar to intravenous injection through oral treatment.Continuous daily administration does not cause system toxicity and has a great anticancer effect.Therefore,NP2 has an excellent prospect for oral drug delivery.