High-gravity technology intensified Knoevenagel condensation-Michael addition po

Xingzheng Liu ,Chuanbo Fu ,Manting Wang ,Jiexin Wang,2 ,Haikui Zou *,Yuan Le, *,Jianfeng Chen,2

1 State Key Laboratory of Organic-Inorganic Composites,Beijing University of Chemical Technology,Beijing 100029,China

2 Research Centre of the Ministry of Education for High Gravity Engineering and Technology,Beijing University of Chemical Technology,Beijing 100029,China

Keywords: High-gravity technology Knoevenagel condensation-Michael addition polymerization Poly (ethylene glycol)-poly (n-butyl cyanoacrylate)Blood-brain barrier Polymerization Reactors

ABSTRACT Poly (ethylene glycol)-poly (n-butyl cyanoacrylate) (PEG-PBCA) is a remarkable drug delivery carrier for permeating blood-brain barrier.In this work,a novel high-gravity procedure was reported to intensify Knoevenagel condensation-Michael addition polymerization of PEG-PBCA.A series of PEG-PBCA containing different block ratios were synthesized with narrow molecular weight distribution of polydispersity indexes less than 1.1.Furthermore,the reaction time reduced 60% compared to conventional stirred tank reactor process.Chemical structures of as-prepared polymers were characterized. In vitro drug delivery performance was evaluated.The cytotoxicity of PEG-PBCA to brain microvessel endothelial cells(BMVEC) decreases with the extension of the PEG chain and the shortening of the PBCA chain.The polymer cellular uptake to BMVECs was better after improving hydrophilicity by PEG block.Results of bloodbrain barrier permeability demonstrated that medium length of PBCA chain and short PEG chain are favorable for hydrophobic Nile red permeation,while long PEG chain and short PBCA chain are beneficial to delivery water-soluble doxorubicin hydrochloride (Dox).The average apparent permeability coefficient increased 1.7 and 0.25 times than that of raw Nile red and Dox,respectively.High-gravity intensified condensation polymerization should have great potential in brain drug delivery system.

1.Introduction

Current therapies formany central nervous system diseases are hampered due to the unsatisfactory permeability of the drug crossing the blood-brain barrier(BBB),especially brain diseases,such as Alzheimer’s diseases and Parkinson’s diseases [1].This barrier is the chief interface to protect the brain against foreign compounds[2].The endothelia of brain microvessels deliver the oxygen and nutrients to brain,such as glucose,vitamins and minerals.These endothelia contain few pores and possess tight junctions,blocking intercellular and intracellular leakage [3].Meanwhile,the activity of efflux transporters and enzymes make the brain get rid of metabolites,toxins and exogenous[4].This dynamic and adaptable barrier strictly limits the permeation of potential toxic and neuroactive substances and pathogens[5],containing many potential therapeutic and diagnostic compounds working at specific areas in brain [6].

In order to overcome this obstacle,many strategies have been performed,such as tight junction modulations,drug molecule modifications and nanoparticle carriers[7].Due to the high chemical and biological stability,feasibility of incorporating different drugs and the multiple routes of administration,nanoparticles were even more attractive for medical applications [8].Wünschet al.[9] built several lecithin coating nanoparticles to adsorb apolipoprotein E3,which can be considered as a transferable BBB targeting strategy.Wuet al.[10] established nanoparticles and nanofibers targeting the brain microvascular endothelial cells(BMVEC),which showed excellent pharmacological activity in siRNA delivery to the brain.Yamaguchiet al.[11]engineered a cyclic heptapeptide based system to facilitate M13 phages cross BBB,reaching 17.8 times higher concentration than that of the control group.Based on the carrier delivery system,Wanget al.[12]designed a wortmannin loaded liposome auxiliary improving the BBB permeability of a gene delivery carriers.

Although there are many therapies that can cross the BBB,there is still a lot of work to improve curative efficacy.As an effective carrier across the BBB[13],poly butyl cyanoacrylate(PBCA)has useful properties for drug delivery,such as acceptable biocompatibility[14],high drug loading capacity [15] and controllable degradation[16].There are several clinical trials reporting the good tolerance[17].Meanwhile,PBCA nanoparticles (NPs) can easily delivery a wide range of therapeutic agents including small molecular anticancer drugs and bioactive macromolecules [18].PBCA with polysorbate 80 or other surfactants coats has been used to delivery many drugs across the BBB [19–22],leading to a potential use in long-term and efficient brain therapy.PBCA is limited by the phagocytosis of reticuloendothelial system and removal of the systemic circulation[23].PEGylation is a wonderful anti-opsonization strategy which can effectively inhibit the phagocytosis [24].PEG block can reduce the opsonization and activation of the complement system [25] to increase the stability and the blood half-life time [26].As the Food and Drug Administration has approved many PEGylated drugs since 1990s such as pegademase(Adagen®)and PEG-IFN-α-2a,the safety of PEGylation has been proved clinically [27].

The PEG-PCA block polymers are usually synthesized by anion polymerization and Knoevenagel condensation-Michael addition(K-M) reaction.Knoevenagel condensation reaction generates αor β-unsaturated compounds from the active methylene and carbonyl groups under the catalysis of ammonia,amine or carboxylate.Michal addition is a conjugate addition between carbon anion and compounds with α-or β-carbonyl,nitrile or nitro groups.K-M reaction consists of these two successive independent reactions without separating or purifying intermediates,which reduces the energy consumption and solvent wastes [28].Thus,this method offers a cheap and environmental-friendly procedure to transform easily available substrates to specific compounds with multiple stereocenters[29].This K-M reaction for PEG-PBCA is carried out by the polymerization between cyanoacetate and formaldehyde,so the cost of materials and storage of monomers are far superior to those in anionic polymerization.At 1997,Peracchiaet al.[30] firstly synthesized PEG-poly (alkyl cyanoacrylate)(PACA) using K-M reaction by condensing PEG,cyanoacetate and formaldehyde in the presence of dimethylamine.Calvoet al.[31]proved the long-circulating and brain targeting of PEG-PACA prepared by this method.Droumaguetet al.[32] designed a selegiline-or rhodamine B-functionalized PEG-PACA polymers targeting an amyloid-β peptide.

Many PEG-PACAs have been synthesized and served for BBB,but it is not clear how their structures and molecular weights(MW) effect the therapy.TheMWof carriers has a significant effect on the drug release properties[33].Our previous work investigated the relationship between the PBCAMWand its biocompatibility and bioavailability[34],and theMWof PEG also influences biocompatible circulation of polymers [35].Therefore,it is meaningful to study the effect of PEG-PBCA polymerMWon its biological activity.In addition,Knoevenagel condensation is a slow reaction,which require high-performance catalysts or promotion by infrared,ultrasound,microwave or heating [36].Given that efforts have been made to improve this reaction from chemical view,the long reaction time and wide product molecular weight distribution(MWD) are still important obstacles for the industrialization of the reaction.Thus,it is critical to find a suitable process intensification strategy to improve the efficiency and reduce the energy consumption.

As a well-established industrial method of process intensification,high-gravity technology has been proved to benefit the manufacture process [37].The high-gravity environment in rotating packed bed (RPB) was generated by the centrifugal force by highspeed rotating packing.The liquid phase from the inlets is chopped into tiny droplets,fine lines and thin films with the contacting of rotating packing.These provide huge mass transfer surface area and the high-speed internal fluid flows breakthrough the limit of mass transfer and micro mixing [38].RPB has been widely used in many chemical reactions,such as acid gas removal [39],NPs preparation by liquid phase reaction precipitation [40] and catalytic hydrogenation [41].Due to the excellent micro mixing efficiency,RPB was used as a premixing device to obtain a uniform mixture of polymerization systems [42,43].For polymerization intensification,RPB was found as an excellent reactor to synthesize a butyl rubber with narrow MWD [44].Our group also reported RPB-intensified anion polymerization,producing PBCA NPs with narrow MWDs,small particle sizes and high productivity [34].

In this work,we investigate the feasibility of high-gravity intensified K-M reaction to synthesize PEG-PBCA block polymers with short reaction time and narrow MWDs.To our knowledge,this is the first time that the high gravity has been introduced to a condensation polymerization.Besides,the physical and chemical characteristics are carried out,and the biocompatibility of PEG-PBCA is demonstrated.Furthermore,the relationship between the PEGPBCA block ratios and the BBB permeabilities is also discussed in detail.

2.Materials and Methods

2.1.Materials

PEG (with the end groups of methoxy and hydroxyl,average molecular weight=1000 and 1900),cyanoacetic acid,4-dimethylaminopyridine (DMAP),N,N’-dicyclohexylcarbodiimide(DCC) and butyl cyanoacetate was bought from Aladdin Biochemical Technology Co.,Ltd.(Shanghai,China).Another PEG (with the end groups of methoxy and hydroxyl,average molecular weight=5000),formaldehyde solution (37% (mass) in H2O),dimethylamine solution (40% (mass) in H2O) and all organic solutions were purchased from Macklin Biochemical Co.,Ltd.(Shanghai,China).

BMVECs were friendly-sponsored from Peking University School of Pharmaceutical Sciences,which was used to build anin vitroblood-brain barrier cell model.All reagents and consumables at cellular level experiments were purchased from Thermo Fisher Scientific Inc.(USA).

2.2.Synthesis of methoxy-poly (ethylene glycol) cyanoacetate

Methoxy-poly(ethylene glycol)cyanoacetate(PEG CA)was synthesized by the esterification reaction between mPEG and cyanoacetic acid,and the operating conditions were based on the literature [30].The reaction mechanism is shown in Fig.1.DCC was used as dehydrator,and DMAP was catalyzer.In details,PEG(10 mmol),DCC (20 mmol) and a little DMAP were dissolved in 150 ml CH2Cl2and 25 ml ethyl acetate.Then,this solution was poured into an RPB reactor purged with nitrogen.The RPB was started and a CH2Cl2solution containing 20 mmol cyanoacetic acid was dropwise added into the RPB reactor with reflux condensation.After reaction finished,about 50 ml ultrapure water was added to terminate the reaction.The dehydration product of DCC isN,N’-dicyclohexylurea,which is insoluble in CH2Cl2.This suspended solid was filtered out and washed by CH2Cl2.The organic phase of filtrate and detergent solutions were concentrated by rotary evaporation and PEG CA solid was obtained after vacuum drying.

Fig.1.Reaction mechanism of the PEG-PBCA synthesis.

2.3.Synthesis of poly (ethylene glycol)-poly (n-butyl cyanoacrylate)(PEG-PBCA)

The polymerization was carried out in an RPB reactor and operating conditions were modified according to the literature[30].The mechanism is shown in Fig.1,and the operation procedure is shown in Fig.2.More specifically,an ethanol and CH2Cl2(1:1)solution containing 1 mmol PEG CA and specific amount of butyl cyanoacetate were added in an RPB reactor with nitrogen purging.Start the RPB and excess formaldehyde and a little dimethylamine were added.After reaction,the slurry was washed by hydrochloric acid,and the aqueous solution was extracted with CH2Cl2.The solvent was concentrated under reduced pressure,and the residue was completely dried under vacuum.

2.4.Preparation of nanoparticles

PEG-PBCA was used as a carrier to load Nile Red and doxorubicin hydrochloride(Dox) to study the cellular uptake and permeability.All PEG-PBCA nanoparticles were prepared by an antisolvent process.PEG-PBCA and the corresponding marker was dissolved in acetone,and 1% PVP aqueous solution was used as antisolvent.These solutions were mixed by high-gravity technology,which was similar to the previous report[45].PEG-PBCA nanoparticles were obtained by lyophilization.

2.5.Measurement

TheMWwas evaluated by a 1525–2414 chromatographic system (Waters,USA) with three Waters Styragel HT columns.Polystyrene (MW:770–270,000 g∙mol-1) was chosen as standard samples.The mobile phase was tetrahydrofuran,and the sample concentration was about 8 mg∙ml-1.Test data were processed by Breeze 2 software and the results were shown as numberaverage molecular weight (Mn,ave) and weight-average molecular weight (Mw,ave).

The nuclear magnetic resonance(NMR)spectra were characterized by an Avance 400 spectrometer (Bruker,Germany).All samples were dissolved in chloroform-d containing 0.03% tetramethyl silane.

The Fourier transform infrared Spectroscopy (FT-IR) was tested by a VERTEX 70v Spectrometer(Bruker,Germany).A small number of samples was uniformly diluted by KBr,pressed into translucent sheets with a manual tablet machine and tested under vacuum below 0.4 kPa.The base line was tested at the same operating conditions using a pure KBr translucent sheet.

An IL4201 drop shape analyzer (KRUSS,Germany) was used to measure the water contact angle.The powder samples were pressed into a sheet by a tablet machine under 10 MPa,and the waxy samples were manually pressed into wafers.A drop of ultrapure water was added by a micro syringe.The drop shape was recorded when the droplet was roughly stable but no more than 10 s.Every sample was tested at least 5 times,and the left and right angles were both calculated into the final results.

The particle size distributions were measured by a ZS90 dynamic laser scattering equipment (Malvern,UK).Appropriate amounts of the waxy samples were dissolved into ultrapure water.Each sample was detected three times at the angle of 90° and a temperature of 25°C.

2.6.Cellular level study

The cytotoxicity was tested using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)as indicator.BMVEC cells were seed in 96-well plates with a density of 5×103cells/well,and incubated at 37°C for 12 h.Then,add PBS solution with different concentration of PEG-PBCA (5–1000 μg∙ml-1) to the plates (n=6),and the PBS solution without polymers was chosen as a negative control.After incubating for 24 h,MTT solution was added and the cells were further cultured for 4 h.Then,dimethyl sulfoxide was used to replace the culture solution with another 5 min incubating.The absorbance of each well was determined by an MK3 microplate reader(Thermo Fisher,USA)and cell viability was calculated by the following equation.

Fig.2.Schematic of the synthesis of PEG-PBCA block polymers.

Here,the ODexp,ODctrl,ODblankare the optical density of experimental group,non-cells group and negative control group,respectively.The subscript number point out the wavelength(nm)of the absorbance.

The cellular uptake against BMVEC was observed by a TCS SP8 confocal laser scanning microscopy (CLSM) (Laica,Germany).The BMVEC was cultured in 8-well chamber slide at a density of 1×104cells/well.The Nile red labelled PEG-PBCA was added in the well at a Nile red concentration of 1 μg∙ml-1.After washing the cells,use DAPI and DiO to die the cell nucleus and cytomembrane,respectively.Then,the dye was discarded and washed away,and some PBS solution was added to maintain the cell morphology.At last,the chamber slide was observed under CLSM excited with 405,488 and 514 nm lights.

The capacity for PEG-PBCA to permeate the BBB was determined by anin vitrocell model [46].The BMVEC cells were cultured in a 12-well Transwell®chamber with 0.4 μm pores.The plate inserts were pre-treated with gelatin solution and the cells were seeded at a density of about 4×105cells per well.At the cell culture stage,the trans-endothelial electric resistance (TEER) was tested as shown in Supplementary Material Fig.S1.At the beginning of the BBB penetration experiment,the TEER of this model was more than 100 Ω∙cm2.The raw model drug was chosen as the negative control group,and the PBCA carrier was the positive control group.All drug delivery systems were diluted to a proper concentration using D-Hank’s buffer solution and detected by high performance liquid chromatography (HPLC) system to determine their concentration.A 1 ml drug delivery system solution was placed in the upper chamber,and the 2 ml blank D-Hanks buffer solution was added into the lower chamber.At set intervals,0.5 ml liquid from the lower chamber was removed and detected by HPLC system to determine the concentration of the model drug.

In order to eliminate the influence of sample solution concentration on BBB osmotic amount,the average apparent permeability coefficient (Papp) was calculated by follow equation:

Here,A(μg∙cm-2) is the amount of accumulative permeation andt(s)is the length of time corresponding to theA.c0is the initial concentration of the upper chamber,which was detected by HPLC system in similar conditions.

3.Results and Discussion

3.1.Preparation of PEG macro initiators using RPB

In order to regulate the PEG chain length of the PEG-PBCA block polymers,a series of PEG macro initiators with differentMWwere prepared using RPB.A uniform micro mixing is helpful to overcome the steric hindrance to speed up this reaction.Considering the polymerization intensification and energy conservation,the high gravity level was chosen as 182.

Fig.3.Molecular weight distributions of PEG and PEG initiator.

When the end group of the PEG is extended,theMWmust be larger than before.Thus,the MWDs of raw PEG and PEG macroinitiators were tested by GPC to demonstrate the reaction process,which are shown in Fig.3.The expectedMWand GPC measured values are compared in Tables S1 and S2.The deviations between the GPC results and the estimated values come from the instrument error,including the properties andMWof standard sample.Since all deviations are roughly the same including the raw PEG,theMWof all samples are considered as expected.

The chemical structure of PEG macroinitiator was characterized by NMR and FT-IR.The NMR spectra of PEG macroinitiators and raw PEG are shown in Fig.S2.The peaks at around 4.37 are assigned to the methylene at the α position of cyano group,which demonstrates that the hydroxyl end group of the PEG is successfully modified into a cyanoacetate group.Besides,the peaks at 1.30 to 1.90 and 6.72 come from the DCU and DMAP impurities,respectively.Here,these impurities will be further removed in the purification steps in PEG-PBCA preparation.The FT-IR spectra prove the synthesis of PEG macroinitiators from another point,which are shown in Fig.S3.The stretching vibration of cyano group shows at around 2250 cm-1,and the stretching vibration of C=O is in connection with the peaks at around 1750 cm-1.So far,we had the PEG macroinitiators ready,and polymerization would be carried out to synthesize PEG-PBCA block polymers.

This procedure was also replicated in a conventional stirred tank reactor (STR),and a 500 r∙min–1stirring paddle was used to facilitate the reaction.The operating conditions were similar except high gravity level and reaction time.The reaction time in STR was 3 h,which was selected as a short period according to the literature [30,31,47].The products from the two processes were compared in terms of the chemical structure,which are shown in Fig.S4.Both NMR spectra are almost the same,proving that both methods can successfully synthesize PEG macro initiators.However,the RPB process took just 2 h to achieve the same goal of the STR process.

3.2.Synthesis of PEG-PBCA block polymers in RPB

Then,the PEG macroinitiators were reacted with various proportions of butyl cyanoacetate to prepare PEG-PBCA polymers with differentMWand block ratios.The temperature was chosen as 50°C for energy conservation reasons,which is just enough to keep the reaction system in a boiling-reflux state.The high gravity level was also selected as 182.On the basis of high gravity promoting,the boiling-reflux state at this temperature can form some fragile cavities,which can further promote the micro mixing.At an excellent micro mixing system,the formaldehyde and ethyl cyanoacetate are in similar concentrations to conduct the alternate growth.In this way,the self-polymerization of formaldehyde is also well inhibited,otherwise the by-products are not easy to remove.

Fig.4.The molecular weight distribution of PEG-PBCA block polymers.

The MWDs of PEG-PBCA block polymers are shown in Fig.4.Because theMWof PEG macroinitiator is already known from the GPC tests above,we can easily calculate theMWof PBCA block by subtracting theMWof corresponding PEG macroinitiator from the totalMW.In Table 1,we compare the estimatedMWwith the GPC tested one.Obviously,there is a gap between the two numbers and the reason is similar as mentioned above.Thus,we use the deviation between theMWof PEG from manufacture’s instruction and GPC result to revise the actualMWof PEG-PBCA.The calculation method is shown in Supplementary Material.Most deviations are less than 10% ,demonstrating that the synthesis of PEG-PBCA is in accordance with the expectation.

Table 1 The expected molecular weight and GPC tested molecular weight of PEG-PBCA block polymers (unit:g∙mol-1)

The chemical structure of PEG-PBCA block polymers was proved by NMR.We chose the1H NMR spectra of block polymers with three different block ratios to show in Fig.5,and the other spectra are shown in Fig.S5.The peaks at 3.64 are corresponding to the methylene of PEG,and the peaks of CDCl3and TMS appear at 7.27 and 0.00,respectively.Unlike the PEG macroinitiator,the PEG-PBCA has 5 kind of peaks from the PBCA block.The peaks at 0.95 represent the methyl of then-butyl side chains,and the peaks at about 1.45,1.74 and 4.29 are on behalf of the α,β and γ position of the ester group on side chains,respectively.The peaks at around 2.40 are the deputies of methylene on the PBCA main chains.The block ratio of PEG-PBCA production was calculated by the integral data from Fig.S5,and the corresponding calculation method is mentioned in the Supplementary Material.Then,the deviations of the block ratios between the estimate and result are shown in Fig.S6.The deviation is no more than 20% ,which proves that we successfully synthesize a series of PEG-PBCA polymers with specific blockMW.

Fig.5.The 1H and NMR spectra of PEG-PBCA block polymers.

The FT-IR spectra offer another way to demonstrate the chemical structure of PEG-PBCA polymers,which are shown in Fig.6.The peaks representing the methylene and C-O-C on PEG arise at 2890 and 1110 cm-1,respectively,which are similar in PEG macroinitiator spectra.The peaks at 2250 cm-1are on behalf of C≡N and the peaks at 1750 cm-1come from C=O of PBCA block.The peaks at 1380 to 1470 cm-1are corresponding to then-butyl side chains of PBCA block.

Fig.6.The FT-IR spectra of PEG-PBCA block polymers:(a) PEG1k-PBCA200;(b)PEG1k-PBCA500;(c) PEG1k-PBCA1k;(d) PEG2k-PBCA400;(e) PEG2k-PBCA1k;(f) PEG2k-PBCA2k;(g) PEG5k-PBCA1k;(h) PEG5k-PBCA2.5k;(i) PEG5k-PBCA5k.

Fig.7.(a) NMR spectra,(b) molecular weight distributions and (c) aqueous solution size distribution of product from high-gravity process and conventional process.

Fig.8.The water contact angle test of PEG-PBCA block polymers:(a) PBCA;(b) raw PEG;(c) PEG1k-PBCA500;(d) PEG2k-PBCA1k;(e) PEG5k-PBCA2.5k.

The PEG-PBCA was also synthesized by STR process.The operating conditions were similar,while the reaction time in STR was 8 h[30,31,47].The PEG-PBCA from different process was compared in Fig.7.The NMR spectra show similar structure,so both of these methods can synthesize PEG-PBCA.For PEG-PBCA from RPB process,Mn,aveis 2969 g∙mol-1,Mw,aveis 3171 g∙mol-1,and PDI is 1.068,while they are 3015 g∙mol-1,3243 g∙mol-1,1.076 for that from STR process,respectively.There is high similarity inMWand MWD for both PEG-PBCA samples.The average size of RPB sample is (304.6±1.3) nm with a PDI of 0.176,while that of STR sample is (342.3±17.1) nm with a PDI of 0.258.It is obvious that RPB is helpful to getting smaller particles and narrower MWD.Due to uneven mixing in STR,a side reaction of formaldehyde self-polymerizing will produce polyformaldehyde,which adversely affects the MWD.

It could be deduced that high gravity intensifies the K-M reaction for PEG-PBCA in two ways.Firstly,RPB accelerated mixing of butyl cyanoacetate CH2Cl2solution and formaldehyde aqueous solution.The micro mixing time is improved to about 10–4s in high gravity environment [48],which greatly promotes the even and quick occurrence of the reaction.Secondly,this K-M reaction is a sequential reaction,and MWD of polymerization product is affected by the uniformity of the reaction system.In a high gravity equipment,segregation index is less than 0.01 [48],where these two reactions continue smoothly to ensure the narrow MWD of PEG-PBCA.

Therefore,RPB process can synthesize PEG-PBCA with smaller particle size,narrow MWD than that from STR process.More important,the reaction times are reduced much more in RPB process,which can increase production capacity and decrease energy consumption.

3.3.Characterization of PEG-PBCA block polymers

A main reason to modify PBCA with PEG block is the improvement of water solubility.The PEG-PBCA polymers with a block ratio of 2:1 were chosen to test their hydrophilicity by water contact angle test.The positive control group is raw PEG,and thenegative control group is PBCA NPs from our previous work [34].As shown in Fig.8,the contact angle of PEG-PBCA polymers are close to that of raw PEG instead of PBCA NPs,proving the excellent hydrophilicity of PEG-PBCA polymers.As shown in Table 2,there is not much different from the hydrophilicity of PEG-PBCA to that of raw PEG,which indicates the hydrophilicity of PBCA is improved greatly after PEG modification.

Table 2 The water contact angle of PEG-PBCA block polymers

Fig.9.The size distribution of PEG-PBCA aqueous solution.

The PEG-PBCA block polymers can easily re-dissolved in water and self-assembled into micro spheres,whose size distributions are shown in Fig.9,and detailed data are shown in Table S3.The particle size depends on both hydrophobic and hydrophilic block proportion.The effect of PEG block on micelle size is complex.Some work reported the micelle size increased with the increase of PEG block [49],while the opposite phenomenon was also reported [50].In our work,the particle size is between 200 and 400 nm,which can promote BBB transport [51].By selfassembling micelles,the PEG-PBCA can load drugs in the hydrophobic kernel,and help drugs cross the BBB or protect drugs from elimination.

Fig.10.Cytotoxicity of PEG-PBCA against BMVECs (n=6).

3.4.Cellular level study

The cytotoxicity of the PEG-PBCA is shown in Fig.10,and the cytotoxicity of all raw PEG and PEG macroinitiators is shown in Fig.S7.The cytotoxicity of all PEG-PBCA against BMVEC is no less than 90% at 100 μg∙ml-1,proving the acceptable cytotoxicity and biocompatibility of PEG-PBCA.Besides,the cytotoxicity of PEGPBCA has a lot to do with the polymer structure.With the increase of the number of hydrophilic units in this block polymers,its toxicity decreased.In other words,the cells can maintain a high concentration of the polymer,and this effect is manifested as an increase in cell viability.The PBCA block decomposes in the cell,and the degradation products causes damage,especially the formaldehyde.Besides,the ratio of PEG and PBCA blocks also plays an important role.This amphipathic polymer will form many micelles binding to the cell membranes,which leads to the change of membrane permeability [47].TheMWof PEG block also effects the cytotoxicity.The length of PEG changes the hydrophile lipophilic balance,which is reported as a universal relationship for many amphiphilic polymers [52].Thus,the cytotoxicity of PEG-PBCA increases with the prolongation of PBCA block,while decreases with the prolongation of PEG block.

The capacity uptaked by BMVECs is also an important assessment criterion for drug carriers.The PEG2k-PBCA1k,which has the medium length of polymer chains,was chosen as the experimental group to test the cellular uptake of BMVECs.Raw Nile red was the negative control group,and the nanosized Nile red and Nile red loaded PBCA was positive control group.As shown in Fig.11,the cell nuclei,cytomembrane and nanoparticles were dyed into blue,green and red,respectively.When the cells have about the same blue and green fluorescence intensity,the PEG-PBCA group exhibits strongest red fluorescence.In other words,the BMVECs internalize more PEG-PBCA polymers than the others.PBCA has been provedas a good assistant for cellular uptaking [34],and addition of PEG block enhances this effect further.PBCA might be limited by the hydrophobicity,which does not disperse well in water.After modifying with PEG block,its hydrophilicity is significantly improved,which has promising future in drug delivery applications.

PEG-PBCA is considered as an excellent carrier to break the BBB for the drug delivery to brain[53].Thus,we established anin vitroBBB model [54] to test the permeability capacity of PEG-PBCA polymers.The Dox and Nile red were used as water soluble and insoluble model drug in this experiment,respectively.As shown in Fig.12,the PEG-PBCA carrier effectively enhances the permeation of Nile red,while this effect is not much significant for Dox.For Nile red,the permeability of PEG1k-PBCA1kincreases by two times than the raw Nile red.PEG-PBCA with largeMWshows poor permeability,and it is close to the two negative controls in the beginning one hour.A short PEG block benefits the high permeability at the first few minutes,and the long PBCA block display a fast penetration rate in the last two hours.At first,the short PEG chain is able to attach enough proteins recognized by the cell,and PBCA segments protect this delivery system across the cell.In the second half of the experiment,the permeability by endocytosis has been basically saturated,while the efflux effect becomes strong.PBCA block can inhibit this efflux effect,so the polymers with long PBCA block shows advantages in the later stage.For Dox,the permeability is not remarkably improved as that for Nile red.The micelle cores of PEG-PBCA are hydrophobic,so the Doxis easy to leak out.Due to the strong damage of Dox,the BBB significant changes in permeability [55].In summary,the PEG-PBCA polymers can notably improve the BBB permeability of hydrophobic drugs,while is less effective for hydrophilic drugs.

Fig.12.Permeation capacity of PEG-PBCA for in vitro blood-brain barrier established by BMVECs (n=3),model drug:(a) Nile red and (b) doxorubicin.

In order to eliminate the influence of errors in sample preparation on this BBB model,thePappthroughout the duration of this experiment was calculated and shown in Table 3.ThePappof Nile red loaded PEG-PBCA is 1.7 times higher than that of raw dyeand 0.58 larger than that using PBCA carries.The PEG-PBCA carriers only increase 25% and 13% compared with PBCA and raw Dox,respectively.In a word,the PEG-PBCA can observably increase the BBB permeation capacity of Nile red,which has significant application prospects in the brain delivery of hydrophobic drugs.

Table 3 The apparent permeability coefficient Papp (10–6 cm∙s-1) for different carriers

4.Conclusions

An RPB reactor was employed to intensify the K-M reaction for the preparing of PEG-PBCA block polymers with controllableMW.Intensified by high-gravity technology,the reaction time was reduced by 60% compared with conventional STR process,and the MWD was narrow (PDI<1.1).NMR and FT-IR spectra characterize the success of the two reactions.In addition,thein vitroBBB related properties were evaluated.The cytotoxicity to BMVEC is acceptable,which increases with the increase of PEG block and the decrease of PBCA block.Using Nile red as a tracer,PEG-PBCA showed excellent cellular uptake performance and BBB permeability.A short PEG chain and moderate length PBCA chain are conductive to the BBB permeation of Nile red,whosePappis 1.57 times higher than the raw material.The PEG-PBCA also shows improvement for the BBB delivery of Dox with a 0.25 times higherPappthan that of raw Dox.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by National Key Research and Development Program of China (2016YFA0201701).

Supplementary Material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cjche.2021.05.005.