CaP 纳米颗粒一将核酸和蛋白质递送至原代神经元/神经干细胞 | MCE全球云讲坛原创

Neural cell systems.Which dresss many of the longstanding challenges in Euro biologys.And today we'are very pleased to have a doctor.Uan Chao associated professors in Taipei medical university as our guest speaker.Doctor child is a distinguished interdismiininary biologist.His research spans from biochemistry genetics and nanotechnology.He earned his PHD from academia syncas then he was trained extensively in different research groups in Kyoto university.Before he started his independent laboratories at Taipei medical university.And he has over a two decades of research experience.And the multi Dis prestigiousious award such as the amble global investigators award and speaking imittations from leading institutes like um em BL and academia silicons.

Doug child is currently leading a pioneering works on gno dynamics in um.Istence and he's going to show us today so for without further duece and please join me in welcoming DR Chu and Chao.Okay thank you.Thankyou all for attending this talk.First I need to thanks a American express for the invitation and thanks data lands for a introduction.I'm from taan medical university Taiwan uh is my honor to be here in the share my study with you so today I will show you how to use this a very cheap homemade re agent to deliver exogenouss material into primary neural and the neurosstes celle for the cellular and the molecule biology investigations.

Okay for a long time we often mark our progress in science by a improvement of image so as you can see this a first nobel price the physicalcal price of a ninety first was an image price give to the well longing.For his a a discovery of a xy and astonish ability to allow the non invanive detection of internal structure within the human body.So this allows doctor to visualize the bond and the other internal tissue we allow surgery by using this pioneering radiation image technology.A few years later in the a ninety six the nobel prize in phycology and the medicine was award to the a came gougge for his development of a silver ni singing to visualize the morphology of neer celle in the brain.

Over the last time I don't think.Sorry excuse me I don't think the the um it's not shared the screen.我同时一样。Okay so uh.Let me try.是。Is screen.Can you see this one uh yes and starting yes yes okay so do I need to start.Start okay okay yes please yes I I don't think it's a share thing okay.So over the century ah there's a numerous advanced image technology have been honored with the nobel prize to highlight their deeply impact on a science and medicine so this technology including the X ray crystal graph.Electro medical school face country micros score uh m m I and city scanning among others so they have dramatically changed how we see everything from the Chinese at allm and the several structure to the vast a cosmic system.

So uh this breaking through continue to ship how we can explore understand and uh innov way across the discipline so as the a saying goeshost this a singing is believing.So many great a great scientific discovery begin with a a simple observation so the more we can see the more we can explore for the centuries people.People have been attracted by the animal that can grow by themselves showing off the beautiful and the shifting colors since the moment people wonder how can they do that.So it'stay a a mystery for a long time until three nobel prize winner finally quickly called.

In two thousand and a or somemea Martin kfi and ragerian won the nobel prize in chemistry because of their are study on bioominance.Uh for this a a doctors monana the first people uh discovered g a p from jellryf fish and demonstrate is unique ability toit green for reasons under the u v line.And for the doctor a kely he demonstrateted that g a p could be used as a forrion marker in a living sale without need for this additional Co factoror making it as a powerful tool for studying a gene expression protein are.A distribution in the several process.For the DR Roger chain he made a a revolutionary contribution in a for protein engineering for the development of a multi colorter variant of these a a g a p which expand is image possibility to visualize and check several process by multiple color simultaneously.

So because of their study today we not only understand the mechanism of the bio romaninis sense but also turned this a growing magic into the most powerful tool which can detect for reason label protein in organism for gene expression checking and living their image by watching.So now we have such a powerful tool to let us to see the intra cellular and the molecule interaction directly.But it still have a big challenge to deliver this a reporter gene into the sales efficiently especially under the primary culture system.So as you can see this image show to differentff neuron level with a transpeive g a p and enttrary respectively to outline their morphology based on the foresten signal we can obtain the detail morphology change of the neuron under either culture over in uh invival.

Consistent.So this is the first time as we can deliver various for recent gene into different prime newur room in the same culture well ah sequially and then monitor the interacting of a preciips and the po snaptic structure under such a high resolution stand.So today I will show you how to use this a very cheap homemade and the two simple step.Are a method to deliver assogenous material into pre neuron and the s sta celle for these s and the molecule biology investigations.So today a numerousous transaction based on the chemical and the biological approach.

A have been developed to accommodate various experimental condition for the physical method it can deliver a a nuc directly into the CAL plasma or nucrius uh so it is a high intensity a fical force like a a electric PAS Jing Guang.Are Michael injection or a mechanical stress to increase the permiability of a sale member.However this fiscal force can also cause a severe damage of a cell membran and a high mortility after treatment.And moreover this a a high technical skill and the expensive equipment are always required for this physical delivery method which is unfriendly for many laps.For a biological method it's always have a graging delivery efficiency but this a a complex gene construction and higher bio safety level are still the major concern for many levels.

Today this a chemical method still the dominant strategy to deliver various exogenous material into sale it's used the carri molecule Le's can neutrize or IMP partt a positive charge onto the nutriacity.The genetic material from this a complexes with this re molecule for delivery.Which you protect them from this degree.Actually this a chemical transaction are generally fall into the three category into the including this a a liy best polyr and the cosing phosphic technique.For the lippy best transaction a lippp song or kalonic liapy is a a a employed to phone this a complexes with the genetic material.

And the lipo a lippo song masss like a lipo factormy are usually in cappul is a nuclear.Within the a across compartment or a by there of a lip song for the delivery so this this complexity can be update directed by the fusion with the cell membran to release the DNA constra inside the cell.For the kit ionic polymer they are a micro molecule with a multi positively charged group along this a polymer backbone and this molecule from the different countries with the nuclear time.Are when this a a electroal static interaction and this complex will be typically a a a optic by this enddo cyosis and then released into the clea through the endle song escaping mechanism.The catalonic poly bears are convenience tool for this a gene delivery but more cyle toxicity because of these a strong electro static interaction that can disrupt this.

The sial membran.Actually this a caing forceosph a precipitation has been a popular transaction method since it's a intro a introduction in the nineteen seventy three uh because it's require only uh easilyas available and UN expensive component the principle of a caosing phosphic delivery involved in this mixing d n a with a caalosing chri in a hippies buffert sin phosph face solution to generate a caing phosph fe d n a cociity.In this Co precipied the caosing FA faith faccilitity is aidding of the condens d n a to the sales surface by eliminating the negative charge of the nuckeine and then promote DNA entering into the celle by enddo syossis.

So this table show you the a comparison among these three different method for the physicalcal method they are a useful for the super difficult transfected sal but may have.Uh may cause this damage and usually lower tissue specific.And for this a bio ah biological method such as a virus are offer high effiency and the long term expression but the high risk of a a bio safetyity imunogennasity and the geneomeic integration are still the concern.And four days are.Method are relatively safe and the a scale but may suffer from this low EI and the trans expression.Actually this a optimal method depends on this experimental design and objective.But in general chemical method is this a optimal choice for most of the experiments.

So today uh this accusing forcements are still a popular transaction strategy because it's a required very cheaper and more convenient component than other chemical method so as you can see this table showed the summary of a conal method for the caosing force BAS a transaction over thirty years many killsing foros BAS transaction tool.Have been developed but the formation of an a optimma killusing for financial particle is difficult to reproduce so it's a a make this a partticle can only be applied in some specific condition and the criteria.We which constraint is a epicability so here you can see the final kills in concentration they use are about two hundred and fifty mini more which is a extremely high concentration ne's can cause the huge kills in phosph precipi.

Just not a nano particle in our system.And recently the formation of a crystal in casing force has been gained attention because of its a applicability in a.A biomedical material for clinical therapy.And importantly recent study has shown this a formation of casing philosophy precipiity does not follow the classical growths mechanisms.For the crystal formation they found a a a small pre nucrireation cluster p n c are the a phone initially and then uh aggregate into the branch polymer assembly.So this is followed by a dissolution and the localized reppiation process uh which gives rise to the a spherical uh domain and the learn to facilitate the formation of a moreous skills in forceos a nano particle a cp.

Ah OK so this a more skills in for a cp show ah.A size in one to five nanno meter in diameter at the early stage then transform into the a twenty to one hundred nanno meter in size eventually so which played a significant role in biomedical and the material science application because of its a.Highbably a highability and a a bio compattibility so unlike other chemical transasion regions such as a cattaonic polymer or lip song caossing forceosphly nano particle show minimal settlele ofx safetyity to make its suitable for a invi in the individual educations.Okay so this table shows the current method for the causing force financialal partic production.

Ah but many limitations such as the high cost a comp protocol and the specialt equipment necessary and the ah disap poor a reproduceability remain a big issue.And the multiple paraymeter show to involve in the formation and the celeability of caosing phosph nano particle.And this a appropriate window of a condition for nano particle formation seems to be narrow so this cause this a major challenge of the low transaction efficiency in this a primary cultural sale.And the recent study has demonstrate this's a formation of accusing forceos fe na no particle can be connectinetically controlled by the cus n to forceosph fe a morear ratio so we try to identify the critical point of the Lisa casing to forceos fe morear ratio for thisaccusing phos nano partic formation.

And under primaryultal systems.So based on the chemical characteristic of a caalosing phosph fe we identify a golden ratio between the caing and the a a phosphway buffer let's facilitate the formation of this accusing forceosph nano particle uh with less than one hundred nano meter in diameter.So this is a a perfect size for this sale update and a extra role cle toxicity.And we perform a systemic study and found that's when the final casing concentration below certified many more.The precipiity can be reduced and.Lisa the a aggregation ability and express this a nano scale science particle.Compared to these fifteen a mini more cert five mini more kills in chri show a similar.A particle size with nano scale but expresss a higher particle number than is fifteen a mini more kills in cor right so indicate that's a certify a mini more casing a concentration create the most significant role in the first step to determine the size of caosing forcely a precipi.

And were a higher production of this a nano particle number within this a window of concentration.So to address the a impact of a different mediadium system on caosing for VI nano particle formation a several common use a luatory mediadium were a investigate in our uh system so we found that's a newural baseel mediadium not only show a perfect a property to generally generally this a small list size but also provide this the highest number of the caosing phosph FA nano particle compared to other media.

So for theses a nano particle inimination we found this h b ss we killing surface best buffer system to remove kills in phos a a nano particle if transfaction.So this data indicate this's a kills in forly nano particle can be generate a con aveniently and simply by this ah using a special mediadium and within a unique killsing to forceosph racial which can be then removed quickly by this accing free h b ss buffer.So we learn a a conductular primary neural culture to review the a transaction effiency on the different skills in concentration and the mediaium system.So the result indicate that's a certify mini more killsing concentration combine with the new base of mediaium system can obtain is the best transaction efficiency.

If we focus on this a map to signal and uh this is a kind of a neural marker it's show that's a caling a concentration below fifty five million more and the using this a a neural bases mediadium can prevent primemi newur room from csyle toxicity during this a gene delivery.And we also found Lisa transion effency also show a positive corre relation with a nano particle number and the Lisa incubation time significantly.Indicated a user can manipate the transaction effency depends on these parameters and a.Are a their experimental purpose so we gave this a strategyeg a new term and the tle faction which combined to whats.A terroror from the Greek and the faction uh is for this a delivery meaning this a a gene delivery method that's is a a t reserved and purpose for design for the goal directed study.

Salvailability was also addressed by this a pstal bre essay under different transaction strategy.And we can see this a tlefe show is a almost no cyle ofx sixty of a primarym neural under fifteen and certified mini more casing concentration and this a under this a neuron vessel a mediad system.And moreover this a neural marker a map two positive sale number was also consistent to this a p pstal bru salvailability as c so t caused is a lower cle toxicity and show no significant difference.To compare with this control group which is a best gene delivery system in.Other methods.

And actually there's a down regulation of enddo sossisis in a mature newural making its poor transaction effency after long term incubation a especially after two weeks culture so we so to uh understand the transion potency of a t faction on different culture day in VI so as you can see this a tlefeion display the highest transaion effency at d I v d I v seven and the start to reduce at d I v eleven gradually.But it still have five and the one point two percent delivery ability at a d I v fifteen and the d I v a thirty a respectively.And the interesting this a number of a gali sale was increased during this a long termm culture but gli sale always show lower transasction e you see after tenle fashionction suggesting this ale fashion is a good tool for a newur a newur a a newurron delivery.

And as combined with this neural specific promoter.So we also compared the tailor function with current popular transion.Transaction kit the data indicate that's a most commercialized kit caused cell death after transaction showing a significant reduction of the a neural marker map to signal.And for this little three thousand and the little masss they show this a lower cycle tox sixtyity than other commercialized kids but tlefe still expressed the best transvasion ability then there a commercialized kit.So uh this flow chart displayed the overview of a causing force financial particle preparation and the process of a tfe so in our system.

This a calsing forceosph nano particle were created by mixing this two more caling chal solution with the two timess concentration of these a h e b s through either these a vtexs or pieting simply without any further incubation.The conditional neural a bassel mediadium from primary neural this is another critical point to generate nano particle so only this the newon basesel mediadium can promote kills in force financialal particle formation.Non newur basedsel mediadium will cause in larger killusing forceosph fe are precipiity and the feel cell death.And after that you will get a.This a tlefection mediadium which include this a conditioned new vessel mediad and casing forri nano particle for the gene delivery.And you just need to replace this a cultural mediadium with this a tlefaure mediadium and the incuba for the desire time followed by a calcium free h b s s wash to finish all the all the process.

So the me questionation is how can we use this talefe for advances a scientific study so here you can see this a this is a a conventional method usually are costs in large causing phosph precipiity and result result in thisapproval transasction evaency and cell deaths.However this a a tlefe method always up to nano skill a.Are kills forly nano particle for higher transfesion efficiency.So as you can see this accusing for vien nano particle show a a a vigorous brownian ma uh uh motion however this a conventional a method usually created this enlarged caosing force sleep precied.

Okay and moreover this tlefeion can deliver this a d n a r a and the protein into the primem newur room and the Spain celle which can be conducted several application.Including this a newural morology examination molecule and the biochemistry analysis.Evacy Co transaction and never neural comparison and this a real time monitoring of a s selecttic connection and the subidial distribution of this for reasonon protein.So terfe can provide a friendly use and the local supporticle without any specialial skill for this molecule and serialal biological biological study in primary neural systems.So for instance.This a terfeion can deliver reporter gene in primary newur room for newural morphology examination so as you can see this is a a fine neural architecture can be illusrate a illustrated by the expression of g p protein after torfe.

So which could be a powerful tool for ah ah examination of this ah fine structural change of a primary neural so as you can see the c can be identified clearly by this uh magenta.Error head and this a blue errorrow indicate xxon terminuss button and this a red errorrow a pointing out this mpase button of the axon and the green errorrow indicate the axon gross com so we can also detect the fine structure of a SPY morphology on this dgetic shift.So in addition study of the molecule and biochemistry change in prime newal with a genetic manipulation is a critical step to uh understand the mechanism of a targetaging.

However this a low d n a trans fashion eanency remain a big issue today so for instance this a important AA is an important approach to investigate this a transnscriptional and translational regulatory mechanism.It's a a still has a a difficulty to deal with thisper neural so here we demonstrate that's ten of affection is a perfect method to a overcome this a long term problem so we found this a tlefeion can provide about million level of a lucifer a a Lu luminiscence intensity.Are from the firefly and readinginna a reciies showing this a similar expression tendency like our previous study.And morever tlefa can deliver d n a and r a for the western blood analysis showing this a significant change protein levelval under the doors and the time dependent minor so this result a a a indicate that's tlefeion is the idea method to deliver nuc into preur room for further molecule and the biochemical study.

And for some a research purpose such as the comunal precipitation and the colonizationation analysis more than to plus me ah more than to plus me a.Will need to be delivered into the sale so in this case tenlefection can provide high efficiency a a Co transaction of a multiple plassy for the exsogenous gene copiration in perurural.So as you can see we make sports the g p and the en chry expression ah plus me for counseling for the nano particle generation followed by this function.

And this a g p positive neuron always call expressed a en chry signal showing this a great cap a capability of the a torfeion for Lisa multiple drink called transaction.So another powerful function of a TOR function is.That it can deliver a various extraordinarygen gene in.To the primary room within the same culture well sequially at a different time points so this'is a allow user to a monitor and the compare the Mo a morphologicalical change of a dream manipululated neuron side by side with a control celle in the same cultural well.So according to this strategy it's a a very interesting we found that's only a rare population of newuron can call express a multiple gene are underquential gene delivery and.

And around ninety.Around ninety percent of these transacted neural a can express only single deliver in after ten affection.So under this strategy we can compare a neural side by side with a different gene expression for instance we deliver in p r one is I r a ah labeled by this m signal first.To not down this inddigegeous uh cm t one expression and this is a a a r a keeping missile transfer for controlling this a dgetic shift of growth.And the two days later g a p plusy was transacted by the tenorfeequially followed by Lia a p f a fixation after forty eight hours incubation so as you can see only this entrary positive newuron loss of indulgenous c t are one signal but not in addressing.

A Cherry negative sale so when we compare the neuron morphology side by side you can see only these are same t one not down neuron showed this the abnormal upization of a dgetic shift.So this is sequential gene delivery porttocol by tlefacturing can let us compare neural side by side within the same cultural well to prevent the biers caused by these are different cultural wills.So in addition a is a real time monitoring of a dynamics my Exxon interaction is still a difficult challenge for the a neural biology field.And a fortunately based on this sequential gene delivery by tlefection dynamic a a visualization in a real time to record either a herotopic SPY xxon or xxon xxon interaction a a a is possible.

Okay so as you can see in this video the pesatticity between SPY and the son was recorded.Let's show this a dynamic in interaction for the s selectpal dusess so based on this method we can conduct its for thisa various studies such as a drug over a gene screening uh uh which involved in this neal genesis and various sale to sale interaction.So a delivery of a esogenouss d n a by this a sequential a tenor affection provided is ideals systemant to investigate a morphologicalical change of control and the adjusting gene target neural ah this can provide these artificial biers or prevent these artificial biers.And the cos by is a different culture well.And the actually detail fact can also a deliver protein into the prime neural since uh this caning forceosph nano particle has been demonsttrated to form the complex with proteining then this a caling proteining particle c p p so we wonder whether the carefeion can generate a caing protein nano particle for the protein delivery in primary neural.

So here you can see in this case the reporter gene can be transacted as.At d I v fifteen of a prem neural followed by this a interesting protein delivery at d I v twenty after twenty four hour the neural will fixed for the further iinal standing and the examination so in this case.Contacting binding protein to c t t n b p two ah with a g p tag.This is.2。Its can by by bitind into this f affecting and the control this a a neural more for genesis so it can be delivered into the sale by telefection and show it's a biological activity by interacting with this a acting stress fiber and a contractive ring.

And this indicate teification is a new strategy to deliver as so joinouss proteining into sale with is a biological activity uh which could be a a an approach to monitor this interaction and the subsarial redistribution of a target p.So in the second case this is a newma a nuclear protein associate with a myto uh myto apperators so this was a they was a conduct to a investigate uh it's a nuclear distribution after tlefe so you can see this's a new ma j p was a successfully transported.Into this nuclearc of this per neal after tlefe so again this demonstrate that'S.

Tfe could deliver as a journals protein into sale and still keep its a biological activity after tfe.So in in addition a a a delivery as a general stream into the a newonte celle ah for study this a Mo a molecule mechanism remain is a significant challenge since t affect has a showed to efficiently a delivered is a nuity and the proteining in PRO neural so we speculate that t fashion may be have this a a a similar capability in the neurossen sales.So as you can see that a transfusion effcy show a positive correlation with the a calsing for nano particle a a loading v and gots higher.

Are gradually when in this a nano particle number increase.And the ten affection also showed this a potential of the proteining delivery into the a neuronstes celle so here you can see this a a protecting by protein to g a p was transacted into the nu DA celle and show a cization pattern with actting streets fiber.And expressed this a normal biological function after tele fashion in neuronste cell.So in this primary newt denial system tenfeion also showed this a great delivery ability of a nuclearcity and the protein which could facilitate the borderer a a comprehension of this molecule and sial analysis.Okay so here we provide this a detail instruction and to enable user to get this high efficient transfasion quickly simply and the Co a cost efficiently uh this can.

Being also enable to apply for the prom newur room and the new Sen cell system are reproducebably so t television will be a for the a a modify and expand to the vival and the envival application so we wish this a a portocol could be a a a foundation for the development of a more advanced delivery strategies.Okay so here we uh appreciate ta medical university and n stc for the funding support and we also uh thanks and for the special gra offering and we are thankful to the professor Huang for her generalrous support all the facility to this a study and I also want to thank the all the people in my lab who work with me to a concrete this a project.

And finally I was deeply appreciate a our collaborator for from this a ball in the in Taiwan for their kind of support OK thank you for your attention and a pressure time.我不想。嗯。I.Yeah I can see that this um techniques is quite can be quite useful for and lots of peoples in the labs because it's a quite common techniques using the chemical wave for and introducing foreigner and genenatic and materials so um.Um I myself have some some questions and first to do do you know how those and I know.Crystals forms is that before you um mixing the denas the genetic materials um or is after you mixing those in a specific buffer and they form those crystals.

And see someone's raiseis in the hands could you please type the questions in the the question and answer box and then so I can pass to the speakers.And.We'have the first question from the audience nice presentations.Would.Patternactionions work for large um plasess or long masssone as and is there an up size limit for efficient transactionions.

Ah yes so in our.Uh we try try to use the different signs of a prosby to to test whether the t a TE fashion has its a limitation and we actually so I have a slide show you here.So.So for your question as you can see here we we we we have tried to deliver a three different press me into the prem newal ah at d v.Are seven and d I v fourteen and we can get a a good results and uh that's all the classmate can be delivered into the primary neural with the perfect expression level and.So uh as demonstrate in this case this a three class me expressed a g p c t r one m Cherry and h a s b f one show this.

A different subsorial localization in nutris a wholesale and the cle solid distribution respectly and they have a a.叫呃呃。Different uh range of the size from the a k b to this the t kbaspaer so that we can successfully call expressed uh in the same prime neural following this a telefe so ah actually tlefeion I think it deliver a a huge size of a classy ah because it can deliver the protein.This protein always they they also have a big new big a big size so big molecule so I think this a ter affection can also deliver this a a.A high molecule.Like a protein or proby into the neural but we tried the a size from this a k b to twelve k b uh within this range but above this range we we we never try before.

So and yeah we have a many questions actually coming in the Chu.Box so the next two questions all regarding the different cell types and for these ah methods and Noah ask ifer is possible to transferact the gleer cells with these matters and zi Xiao asked if is there any potential to apply.This technique to like adherences and a suspension cell I.Uh actually we also uh uh try to use tion in different cell line so in this case.How to say okay so actually t fact ISA.Are a a fundamentally a based on this a killing phosphation.

So it has a long been used for transacting variousers IM motortalize cell line and in a this figure we used to human cell line to na tea and hela sale to test the a transaction efanency of tlefection.And for the two naceity sale so it's a as you know it's a common uh used the human embronia kidney sale line a particularly for the is a gene exploration and viral production so we can see this a television fashion can up a up obtain over ninety percent transfeion effiency under two hour incubation ah with this a cursing forly nano particle.And for this hela sale this is a human cervical cancer sal line it's show about seventy percent transaction effency are under to our causes forcely nano particle in incubsion and actually we also try cost seven sal line this is a a Africa green a a green monkey a kidney sale that that we can have.

About sixty percent transfesion effiency by telefeure so this results showed is a a sal line they are more robust than primary neural that's a terriion protocol can be a a a adapt to this a sal line system with the high ah transion eanency.At least in this two three two nine three t and healer and the cost seven civil lines.Okay and we have a.Many more questions coming and and they ask him.Ah he's wondering if whether the nano calcium particles can um.Immediate deliver degreeing for it this different as system is one hundred percent reprodduceable and how long you can keep this um form forming the nonno partlyles.OKSO.

Uh I also prepare this table so.呃。Okay so not this one show you another one.So how long this region can be stored.Okay yeah OK so uh actually we try uh to test how.How long?Ah for this a tfe region can beto so ah.Actually thiss a PH h PH h values ah stability of this a two X h he b s is very critical issue to produce this causing philosophy ah not all particle so we uh try to understand how stable is about this two X h e b s on the different.Conditions so as you can see this a up left table we demonstrate that's a this a two X h e b s is a super sta under this a harsh condition that's all can give a good gene delivery into the pre neal and the blow table ah showed this a a a.

A storage condition in the stability during a a Du of the tle affection region religion in the buffer so as you can see a exception of the telefeion master mix should be prepared and the use freshly.And for the other television religion and the buffer that are very stable and can be s store on the regular condition for a long time.And Anthony also asked there's a size of the protein and the fact of the telefas efficiency.Oh yes we try different sizes protein ah including just show you before newma and the city by the proteinting too and we also try this a a antibody so the size we try is a from the.

Twenty uh kill downton to almost one hundred kill downton so within this range I think this no problem but we found as this protein size become bigger the nano particle size will uh slightly.A become a enlarged so in this case I try uh I I I suggest uh uh the user it's better to uh reduce this accusing phosphic concentration to reduce this a uh.A particle size.嗯。So the next question um asking about the um different cell types um yeah so um have you have you tried to apply these telefes into other primary cell cellize such as um the primary havep to havep to side.

Okay and this's a good question because I also s studydied the a liver so I we in our level we also prepare this a primary partan culture so I tried this a televisionfe.Delivery in the promulure system but this a a.Be honest the eff is not so good.For the prosin delivery.Uh it's maybe one to two percent this is very lower so uh but for this side we found for this protein delivery it has very high transssion a high a a a yeah high transssion delivery it's a maybe have this almost ninety percent protein delivery.This protein is an antibody for recent country antibody so it's have almost ninety percent a protein delivery.Ah because we can detect this for signal uh depradation and we found this a protein can be updateted by this hypoolide preulture hypoolide but they will be degree uh within me.

Uh maybe forty eight hour the health health life maybe be a forty hour so the problem I think the problem is a this sale can update.The or protein but thisim or protein may will be degree.During the escape.These are andson.啊，看啊。A complex so maybe a one problem for this hypool SE herric cultureture we need to do is to prevent this uh enddo sideossis.DNA or protein deradation this may this maybe will a a a improve this a transaition effency.Um I can see in the audience there many people doing ah ste cell research and as I can see many questions asking um have you tried um this matters on like embryonic stand cells and yoururative cells or and indduce the prop PRO protein and ste cells.

Before differentiation yeah.Yeah yeah yeah.So uh OK this this is why I show you before actually we also try to use the.A transion events showed this a positive relation with a csing enforcement na particle it's a will ah ah when you increase this a csing forcely naal particle number this a transion sale number will increase so we we we we have this a a good transion efancy under this two hundred microor uh a televisionerlefe media.And I think for the neuronsens cell is'no problem for the tfeion to deliver this a dream a plus me and the protein but we never try all kind of a ste cell.

And yeah I can see and subabrino is raiseising a hand in the audience could you please ah type your questions into the question I answer box please.Um yeah the next question from jumping and.'asking on the other side of these ah process is ah um can you apply this techniques with the message on a the stable with the tele facts.OKOK.We also try to use t fashion to deliver m r a ah it's a m can be delivered by tenfeion but in our case maybe this we cannot handle this amount a perfect.So it'has I think it has some degradation so compared to this uh uh.DNA delivery m RNA delivery is I think it's a lower.

A l a plusmate.Yeah I think the PRO major problem is the.A stability of mon not for this tlefe itself.嗯。The next one is from Eric um so.So he ask it seems to have a low toxicity um than other commercial kit does it has any side effect for the neurons during the tele facts procedures.Okay does the cells have any response or change during the teleifications.Okay you put procedures.Okay so ah okay so this data we we we show in our supplementary data so during this parafeion we also try to ah.To monitor this a cell phology.

And some a a sale activity ah by this accusing image and electoral phology.So here you can see this uh the efficy of a cursing forcely nano particle a.Ah the in fact of the this kills in enforce financial nano particle on on this electal phology of the primary neural a show as this a line graph ah okay so this is a a kind of a sodium channel chasing a with is a ivv curve so the data indicate during a telefe neuron show a around forty percent a down regulation of a sodium current.But it's still or but but but this a a a inhibition was rescued by a replaced the terfeion mediadium with the h b s s or conditional mediad.And for the a casing image we found this intraial uh.

A killusing was increased about ten percent and showed a it has a a statistic significance but this ten percent killsing elevation did not impair n d a media neural activity so ah actually.A tenfeion show only mind in fact on this a newuron a phsology during a nano particle incubation so uh uh this can be revered this is reversible after releasing from this a affectual media.So so television has in fact but not so strong and it is revers.Right and we would take two more questions okay and yeah so this John's come back and he asked him.And is the calcium phosphate complex it um escaped from the anders while the low PH Anderson liseron transitions.

And how would you think about the erratio um of the population that successfully get to the cycless songms and then to the NUS.Yeah I think this'is good good question but we have we don't have answer right now and we try to use a different.Different ratio and the try to use different ah enhibitor or a a a indur to enhance this transaion effiency but it's still undergoing so uh.It's a difficult for me to answer this question sorry.So um I think it'the lot the last question just ask you to maybe highlight the um the this specific advantage of using the councilciosph nanopolycules.嗯。

What's the specific advantage?Okay I thinkion is very cheap and you can.This is a homemade region you can use very uh a cheap kills in force fe and prepare older region in your lab you don't need to buy this little fatantamming or other commercialized kit.And I think this's a for all the neuron it's a lower cle toxic a.A compared to this a control group so ah I think it is a good tool for the especially for the preureal culture for the gene delivery but people need to to know uh it's very a critical information I show you here you need to know uh uh uh as you can see this table show the different uh.Are a difference between the this a new neural basedsal mediadium and the conditional mediadium so these are very important when you translate.

But when you use t function to translate this appur plasmate into a primem newur room we strongly to a suggest you you have to use this conditional nu baseds media you cannot use the new nuur baseds media because these a the ionic balance autosmotic uh uh or uh athmoarity and multipleabize will be changed after several death of a so culture so this uh athmotic and ionic shockrk may be occur.When a switching from this condition mediadium to the fresh neural bestel mediadium in per neural okay because of change of these multiple multiple subtle but important factor for the iron balance and it's may cause this a cle toxicity so when you use this a t of faction please remember.Use these are conditional neuron vessel mediad do not use the new one but when you when you apply.

Into this.A sal line system that's no problem you can use the new new bases media.And thank you very much drchwman Chao for such insightiful presentation today and you ah thank you to all the audience members for joining us for this websiteards.Their more exciting macin express webs coming up ah very student um our next confirmed um webs were focus on the steroids receptor singings in breast cancer so um please stay in tune for that.And and American express we'are committed to fostering um the scientific communications and accelerating the pace of a scientific discoverings we thank you again for joining us today's and the say connected.And I look forward to seeing you all.