CN117442583A - Self-assembled PROTAC peptide nano-carrier and application thereof - Google Patents

Abstract

The invention provides a self-assembled PROTAC peptide nano-carrier, which is characterized in that: PROTACs are loaded into neurotransmitter-like nano-carrier NPs by utilizing calf thymus DNA intercalation technology to prepare PROTAC-loaded nano-particles, and the PROTAC-loaded nano-particles are applied to preparing medicines for treating Alzheimer's disease or relieving Alzheimer's disease symptoms.

Description

Self-assembled PROTAC peptide nano-carrier and application thereof

Technical Field

The invention relates to biotechnology, in particular to a self-assembled PROTAC peptide nano-carrier and application thereof.

Background

Alzheimer's Disease (AD) is a refractory neurodegenerative disease characterized by amyloid plaques and tau tangles, the main symptoms of which are memory loss and progressive neurocognitive dysfunction. The main pathological features of AD are senile plaques and neurofibrillary tangles (NFT), consisting of extracellular β -amyloid (aβ) and intra-neuronal hyperphosphorylated tau, respectively. However, to date, many "promising" drug candidates for aβ -related pathology have failed in clinical trials. Thus, other alternative targets for AD treatment may be considered at the time.

tau protein is an important microtubule-associated protein that acts as a stabilizer for neuronal microtubules. When tau is hyperphosphorylated, it migrates from microtubules and triggers the self-proliferative cascade of tau aggregation, leading to toxic NFT production, neuronal cell death, and ultimately AD. Since tau is a key driver and potential target for AD, targeted clearance of tau may be an effective means of treating AD.

The protein degradation targeting chimeras (PROTACs) technology is a fast and efficient novel target protein clearance strategy. The protoc technology has some limitations at the same time:

(1) the PROTAC drug has certain toxic and side effects: proTAC can thoroughly degrade pathogenic proteins (such as hyperphosphorylated tau protein), but can accidentally injure normal proteins of the body (such as tau protein involved in maintaining cytoskeletal stability);

(2) the stability and half-life of PROTAC drugs in vivo are to be improved;

(3) the profacs have difficulty treating AD across the blood brain barrier: due to the existence of blood brain barrier, PROTACs are difficult to effectively enter brain tissues to play a role, and after entering brain, enrichment in lesion sites (cerebral cortex and hippocampus) is difficult to achieve effective treatment concentration;

(4) the degradation of high polymers by ubiquitin-proteasomes by procac drugs is inefficient, especially in diseases where the ubiquitin-proteasome pathway is dysfunctional (e.g. AD).

Thus, it is currently difficult for PROTACs to effectively exert the desired therapeutic effect on AD.

Disclosure of Invention

The present invention aims to overcome the above drawbacks and achieve the aim of treating AD by using neurotransmitter-like carrier (NPs) to carry the PROTAC molecules to achieve penetration of the blood brain barrier and clearance of tau proteins overexpressed in the brain.

The invention provides a self-assembled PROTAC peptide nano-carrier, which is characterized in that: and utilizing the negatively charged intermediate to load the PROTACs into neurotransmitter-like nano-carrier NPs by using an intercalation technology so as to prepare the PROTAC-loaded nano-particles.

The intermediate with negative charges can adsorb blank nano carriers NPs and PROTAC peptides with positive charges in the self-assembled PROTAC peptide nano carrier in a manner of electrostatic adsorption with positive charges, and the blank nano carriers NPs and PROTAC peptides with positive charges adsorb the former two to form stable mixed nano particles. Alternatively, calf thymus DNA having this property, and other small molecules having similar functions.

Further, the self-assembled PROTAC peptide nano-carrier provided by the invention is further characterized in that:

the neurotransmitter-like nano carrier NPs are prepared from one or more mixed lipids/lipoid selected from NT1-O12B, NT1-O14B, NT1-O16B and PBA-Q76-O16B.

In the preparation process, the two substances are respectively measured according to a certain mass ratio, dissolved by absolute ethyl alcohol, fully mixed, added into a dispersing solvent (sodium acetate solution) dropwise, and subjected to standing to self-assemble to form uniformly dispersed Nano Particles (NPs).

Further, the self-assembled PROTAC peptide nano-carrier provided by the invention is further characterized in that:

the mass ratio of the NT1-O16B to the PBA-Q76-O16B is 0-7:3-10.

Further, the self-assembled PROTAC peptide nano-carrier provided by the invention is further characterized in that:

the preparation method of neurotransmitter-like nano-carrier NPs comprises the following steps:

s1, dissolving NT1-O16B and PBA-Q76-O16B in alcohol;

s2, dropwise adding the mixed solution of the S1 into a sodium acetate solution, and then gently oscillating;

s3, dialyzing, and collecting the mixed lipid solution.

Further, the self-assembled PROTAC peptide nano-carrier provided by the invention is further characterized in that:

s4, adding PROTAC molecules and UPH2O into the mixed lipid solution of neurotransmitter-like nano carrier NPs, and standing at room temperature for 5-20min;

s5, adding UPH2O into calf thymus DNA, and standing at room temperature for 5-20min;

s6, dropwise adding the S5 into the S4, repeatedly blowing for a plurality of times, and standing at room temperature for 5-20min to obtain the PROTACs-NPs nano preparation.

Wherein the weight ratio of the mixed lipid to the PROTAC molecule to the calf thymus DNA is 2-6:1:0.1-0.5 (optimal 4:1:0.2), the rotating speed is 300-600rpm, the dropping speed of the microinjection pump is 5-10ml/min, and the hydration is 10-50min.

In addition, the invention also provides application of the self-assembled PROTAC peptide nano-carrier in preparing a medicament for treating Alzheimer's disease or relieving Alzheimer's disease symptoms.

In addition, the invention also provides application of the self-assembled PROTAC peptide nano-carrier in preparing a medicament for treating or relieving ubiquitin-proteasome pathway dysfunction.

In addition, the invention also provides application of the self-assembled PROTAC peptide nano-carrier in preparing a medicament for improving AD cognitive behavioral functions.

Further, with respect to the above application, it is further characterized by:

the effective dosage of the drug, self-assembled PROTAC peptide nano-carrier is not higher than 20 mu M.

In addition, the invention also provides application of the self-assembled PROTAC peptide nano-carrier in preparing a preparation or a medicine for removing over-expressed pathogenic protein p-tau protein.

In addition, the invention also provides an AD cell model, which is characterized in that: the plasmid overexpressing GFP-tau was transfected into N2a cells.

The invention has the following functions and effects:

in the research of the invention, the related verification experiment proves that the PROTACs-NPs nano particles have good particle size, uniformity and stability;

The PROTACs-NPs nano particles can effectively induce tau protein over-expressed in cells to be cleared in vivo and in vitro;

the ubiquitin-protease system participates in the clearance process of the over-expressed tau in the cells mediated by PROTACs-NPs, and the NPs serve as nano carriers to effectively play a role of a nano delivery system for targeting cells and brain lesion sites, so that the effect of the PROTAC molecules in treating AD in vivo is ensured;

and the PROTACs-NPs nano particles have good biocompatibility in vivo.

Drawings

FIG. 1 NPs nanofabrication appearance and particle size at different ratios of NT1-O16B and PBA-Q76-O16B formulations;

FIG. 2. Scan results of NT1-O16B and PBA-Q76-O16B under Jeol 400MHz nuclear magnetic resonance spectrometer;

FIG. 3. Molecular sequence of synthetic TAU-PROTAC peptide;

FIG. 4. Preparation of PROTACs-NPs nanoformulation;

FIG. 5 characterization of NPs and PROTACs-NPs (Size, particle Size; PDI, polydispersity index; TEM image, projection electron microscope image);

FIG. 6 in vitro storage stability and in vivo stability of PROTACs-NPs nanoparticles;

FIG. 7 shows the drug release profile of PROTACs-NPs in PBS at 37℃or PBS containing 10% FBS;

fig. 8. Protas-NPs cytotoxicity against N2a experiment (n=5);

FIG. 9 fluorescence intensity of in vitro BBB model transwell indoor medium detected by enzyme-labeled instrument;

FIG. 10 fluorescence of N2a cells in the in vitro BBB model transwell chamber under a fluorescence microscope;

FIG. 11 WesternBlot experiment results of PROTAC and PROTACs-NPs inducing tau clearance in N2a cells at different drug concentrations;

FIG. 12 Co-immunoprecipitation (Co-IP) of endogenous Keap1 and tau (55-70 kDa);

FIG. 13 flow cytometry detection results of PROTACs-NPs and PROTACs inducing tau clearance in N2a cells at different time of action;

FIG. 14 is a fluorescence imaging diagram of an isolated organ of a mouse after 2h of drug treatment and fluorescence intensity quantitative analysis thereof;

FIG. 15 is a fluorescence imaging diagram of the mouse ex vivo brain after 2h of drug treatment and fluorescence intensity quantitative analysis thereof;

FIG. 16 fluorescence of mouse brain frozen sections after 2h of administration of Cy5-PROTACs-NPs, cy5-PROTAC, PBS;

FIG. 17. Sequence of water entry positions of mice during the training phase of the water maze;

fig. 18 cognitive behavioral assessment following treatment of 3 xtg AD model mice with TAU-PROTACs-NPs, (n=6 per group), <0.01, <0.001;

tau-PROTACs-NPs clear brain focal tau protein in vivo: (A) And (C) represents AD mouse cortex and sea horse brain tissue protein sample to carry out Western Blot experiment various protein (Tau, p-Tau, keap1, beta-actin) band diagram; (B) And (D) represent the normalized gray scale ratio of p-Tau/beta-actin in (A) and (C), respectively. (n=3 per group) & lt 0.01, & lt 0.001;

FIG. 20 detection of the expression level of phosphorylated tau protein in brain tissue sections (cortex, hippocampus) of 3 XTg AD mice by immunofluorescent staining after drug treatment: (A) Immunofluorescence sections of cortical and hippocampal areas of AD mouse brain; (B) And (C) is a bar graph normalized by the ratio of pTau (Thr 205)/DAPI fluorescence intensity in (A). (n=3 per group) & lt 0.05, & lt 0.01, & lt 0.001.Scale bars are 200 μm and 20 μm;

fig. 21, weight change (n=6 per group) during treatment for mice of each treatment group;

fig. 22 comparison of the vital organ weight coefficients of mice of each experimental group (n=6 per group)..p <0.05;

fig. 23 blood biochemical indicators, including liver function indicators (ALT, AST, and ALP) and kidney function indicators (urea, UA, and Cr) (n=6);

FIG. 24 shows the results of HE staining of pathological sections of vital organs of mice in each group after treatment, scale bars are 200 μm;

FIG. 25 Structure and characterization of NT1-O16B lipids and PBA-Q76-O16B;

Detailed Description

Example 1 preparation and characterization of PROTAC-loaded neurotransmitter-like nanoparticles (PROTACs-NPs)

1.1. Preparation of PROTAC-loaded neurotransmitter-like nanoparticles (PROTACs-NPs)

Measuring 300 mu gNT of lipid and 700 mu g of lipid PBA-Q76-O16B (structure and characterization are shown in figure 25) (total amount is 1mg, wherein the weight ratio of NT1-O16B to PBA-Q76-O16B is 0-7:3-10, as shown in figure 1, NPs obtained at the ratio of 3:7 have optimal nano particle size and the external transparency of the nano particle solution is optimal), and dissolving in 100 mu l of ethanol; 100. Mu.l of sodium acetate solution (25 mM, pH 5.2) was prepared, 100. Mu.l of the mixed lipid solution was added dropwise to 300. Mu.l of sodium acetate solution (25 mM, pH 5.2), followed by gentle shaking; adding the mixed solution into a dialysis bag with a molecular weight cut-off of 35kDa, dialyzing MilliQ water for 12 hours (1 time for 6 hours, and the total two times), treating the dialysis bag after the completion of the dialysis, and collecting a sample. The mixed lipid solution, PROTAC molecules (synthetic route is shown in figure 3, amino acid sequence: N-terminal-YQQYQDATADEQG-GSGS-LDPETGEYL-RRRRRRR-C-terminal) and calf thymus DNA (Sigma-Aldrich Co., U.S.) are incubated together and self-assembled to form nano-liposome (the weight ratio of the mixed lipid, PROTAC molecules and calf thymus DNA is 2-6:1:0.1-0.5, optimal 4:1:0.2, rotational speed is 300-600rpm, the dripping speed of a microinjection pump is 5-10ml/min, and hydration is 10-50 min): taking 124 μl of mixed lipid solution, adding 15 μl PROTAC molecule (peptide 1, concentration of 2 μg/μl), adding UPH2O11 μl, and standing at room temperature for 10min; taking 2.4 mu l (concentration is 10 mu g/. Mu.l) of calf thymus DNA, adding UPH2O57.6 mu l, and standing at room temperature for 10min; dropwise adding calf thymus DNA solution into the mixed lipid/peptide 1 complex solution, repeatedly blowing for several times, and standing at room temperature for 15min to obtain PROTACs-NPs nanometer preparation (synthetic route is shown in figure 4).

NT1-O16B lipid and PBA-Q76-O16B lipid are used

MaF, yangL, sunZ, et al, neurotransmitter-derivedlipidoids (NT-lipidoids) for enhancement of dbracindedulcithrough interlineausinginjections, sciAdv 2020;6 (30) eabb4429. Publichod 2020jul24. The nuclear magnetic results are shown in figure 2.

1.2. Characterization of PROTAC-loaded neurotransmitter-like nanoparticles (PROTACs-NPs)

The particle size, PDI and potential of the prepared PROTACs-NPs nanoparticles were measured by a Markov particle size meter, and then the morphology of the nanoparticles was observed by a transmission electron microscope. The results are shown in FIG. 5, which demonstrates that the PROTACs molecules successfully load into NPs and self-assemble into spherical liposome-like structures, with a Zeta potential average of about 50 mV. The results show that the PROTACs-NPs nano particles prepared in the embodiment have smaller particle size, better uniformity and easier permeation through cell membranes and blood brain barriers.

1.3. Stability test

As shown in fig. 6, the protas-NPs nanoparticles remained stable in PBS solution at 4 ℃ (simulating in vitro storage conditions) or in PBS containing 10% fbs at 37 ℃ (simulating in vivo environment);

as shown in fig. 7, the protas-NPs stably sustained release protas in PBS at 37 ℃ or PBS containing 10% fbs.

The PROTACs-NPs nano particles prepared by the embodiment have good stability and the property of slowly releasing PROTAC molecules.

Cytotoxicity experiments of ProTACs-NPs nanoformulations

1.4.1 configuration of the medium: formulation of N2a cell complete medium: 45mL of high-sugar medium (DEME) +5mL of Fetal Bovine Serum (FBS) +500. Mu.L of penicillin-streptomycin diabody solution (P/S). In a clean bench operation, 45ml of DMEM medium was poured into a 50ml centrifuge tube, 5ml of FBS and 50. Mu.l of P/S solution were added thereto, and after thoroughly mixing, DMEM medium containing 10% FBS and 0.1% P/S was prepared.

Resuscitation of n2a cells: preparing a water bath kettle containing a certain amount of clean water, heating and adjusting the water temperature to about 37 ℃, then rapidly taking out a freezing tube in which N2a cells are frozen from a liquid nitrogen bottle, putting the freezing tube into the water bath kettle, and repeatedly stirring the freezing tube with tweezers to enable the freezing tube to be melted rapidly. After complete thawing of the frozen stock, the cell suspension was transferred to a 15ml centrifuge tube (5 ml fresh medium added in advance) in a sterile operating table. The supernatant was then centrifuged (1000 rpm,5 min), carefully aspirated, 1ml of complete medium was added and resuspended cells were gently blown and transferred to 25cm2 flasks with 4ml of complete medium added beforehand, gently shaken to disperse the cells evenly, and finally the flasks were placed in a cell incubator (5% CO2, 37 ℃).

Culture and passaging of n2a cells: when the cells are in logarithmic growth phase and the cell confluence reaches about 70-80%, the cells can be passaged. The old culture medium is sucked and removed, 2mL of PBS is added, the PBS is gently shaken and then sucked and removed, 1mL of pancreatin is added after the washing is carried out for 2 times (the bottle body can be properly shaken to enable the pancreatin to fully contact cells at the bottom of the bottle), the old culture medium is placed in a cell culture box for digestion for 1min and then taken out, the cells are seen to flow down in a sediment manner when the culture bottle is inclined, and 2-3mL of complete culture medium is added to stop the digestion. The non-detached cells on the flask wall were repeatedly blown off with a 1ml pipette to completely detach the cells from the flask wall, collected into a 15ml centrifuge tube, leveled, and centrifuged (1000 rpm,5 min). Then the centrifuge tube was removed, the supernatant was aspirated, 1ml of complete medium was added to resuspend the cells, and the mixture was blown down and mixed well according to 1:2 or more, and supplementing the culture medium to 5ml, and uniformly mixing by a cross shaking method. Finally, the alcohol sterilized cotton balls are used for wiping the sterilized bottle body, the bottle body is prevented from contacting the bottle mouth part, and the bottle body is placed in an incubator for continuous culture. Note that: because N2a cells belong to semi-adherent cells, the damage is caused by the too long digestion of pancreatin, the digestion time (about 1 min) should be controlled, and the digestion is stopped in time.

1.4.4. Cell count: n2a cells were digested by pancreatin, and after termination of digestion and centrifugation, they were diluted several times with complete medium depending on the number of cells and the growth state. Transferring the cell suspension into a cell counting plate, taking care to avoid generating bubbles, carefully inserting the counting plate into a cell counter to perform measurement reading, and properly adjusting the cell suspension according to the measured cell density so that the density and the number of cells meet the experimental requirements.

Cryopreservation of n2a cells: n2a cells in the logarithmic growth phase were selected, after washing the cells 2 times with PBS, digestion was terminated with pancreatin, and the supernatant was discarded by centrifugation (note: the above steps were the same as the cell passaging process). 2ml of cell cryopreservation liquid is added into a 15ml centrifuge tube, cells are resuspended, and after the cell suspension is gently blown and evenly mixed by a pipetting gun, the cell suspension is averagely transferred into 2 cell cryopreservation tubes, the cryopreservation tubes are covered, and cell names and cryopreservation dates are marked. And (3) placing the freezing tube in a system cooling box, placing the freezing tube in a refrigerator at the temperature of minus 80 ℃ overnight, and finally transferring the freezing tube into liquid nitrogen for preservation. After 2-3d of cryopreservation, a tube of cells can be randomly withdrawn for resuscitation to check the cryopreservation effect.

Cytotoxicity assay of ProTACs-NPs nanoformulation: the cytotoxicity of the PROTACs-NPs nano-preparation on N2a cells is determined by adopting a CCK-8 method. (1) cell plating: taking N2a cells in logarithmic growth phase, digesting with pancreatin for 1min, stopping digestion, collecting, centrifuging, and discarding supernatant. Cells were resuspended in complete medium (DMEM with 10% FBS and 1% P/S) and counted to adjust the cell density to 5X 10 ⁴ Each 96-well plate was inoculated one by one at a volume of 100. Mu.L/well, at which time a set of blanks (without fine inoculation)Cells). To prevent the edge effect, 100 μlpbs was added to the peripheral round of each set of multiple wells. The cell plates were then placed in a 37 ℃ cell incubator overnight to allow cell attachment growth. (2) preparation of PROTACs-NPs nano preparation solution: first, a mother solution of the PROTACs-NPs nano-preparation (with a concentration of 287. Mu.M) was prepared according to the above method, then diluted to 60, 50, 40, 30, 20, 10. Mu.M with the above complete medium, the nano-preparation was filtered with a sterile filter sieve of 0.22. Mu.M, and finally stored in a refrigerator at 4℃for use. (3) After incubating the cells with PROTACs-NPs, cell viability was measured, the N2a cells with good adherent growth were removed from the incubator, old medium was aspirated, and the cells were washed 2 times with 100. Mu.LPBS. Then, according to 5 compound wells of each concentration group, 100. Mu.L of the above-mentioned medium containing PROTACs-NPs at different concentrations was added to each well, and only the complete medium was added to the final compound well group (as untreated group). After the addition, placing the 96-well plate in a cell incubator for continuous culture and incubation for 24 hours, sucking the old culture medium, washing the culture medium for 2 times by using PBS, then adding 100 mu L of CCK-8 culture medium containing 10% into each well, continuously placing the culture medium in the cell incubator for culturing for 1 hour, and taking out the 96-well plate. The 96-well plate was placed on an enzyme-labeled instrument and oscillated for 15s to facilitate sufficient mixing, then the OD value of each well at a wavelength of 450nm was detected, cell viability was calculated according to the following formula and a cell viability graph was made. Cell viability (%) = (experimental OD value-blank OD value)/(untreated OD value-blank OD value) ×100%.

As shown in FIG. 8, the cytotoxicity test results show that when the concentration of PROTACs-NPs is less than or equal to 20 mu M, the survival rate of the cells is more than 80% after the PROTACs-NPs are incubated for 24 hours, and the cells have no obvious toxic effect on the N2a cells. According to the experimental result, PROTACs-NPs with the concentration of 20 mu M or below are selected for subsequent experiments, so that the PROTACs-NPs can not generate obvious toxic or side effect on N2a cells while generating tau protein clearing effect.

Example 2 ProTACs-NPs nanoformulations induce inflammation of tau clearance in tau-overexpressing N2a cells

Uptake of fluorescein-labeled PROTACs-NPs nanoformulations by N2a cells

1. Type I rat tail collagen was thawed in a refrigerator at 4℃for 1h in advance and transferred to an ice chest prior to the experiment.

2. The 1.5mLEP tube, transwell chamber and tip were placed on ice for pre-cooling.

3. According to the experimental use amount, the rat tail collagen type I is diluted into a use solution on ice according to the concentration recommended by the specification, and the three-dimensional collagen is prepared. (see I type rat tail collagen instructions for use)

4. After cutting off a small section (about 3 μm) of the gun head, a certain amount of collagen diluent is sucked on ice, lightly added into the upper chamber of the transwell chamber, and the liquid is spread on the bottom to ensure that the collagen solution is spread on the surface of the vessel, and uncapped in a super clean bench for overnight air drying. Or it can be used directly after being left at room temperature for 1 hour and washed 3 times with PBS.

5. The cell climbing sheets with the size of 48 pore plates, which are used as required, are soaked in 75% alcohol for 15min and then placed in 24 pore plates, and can be used after being irradiated by ultraviolet rays of an ultra-clean bench for 30min, so that no alcohol exists in the 24 pore plates.

6. After digestion, centrifugation and cell counting (bEnd.3cells), the cells were diluted with serum-free medium to a concentration of 2.5x10 ⁶ Cell suspension per mL.

7. According to 100-200 mu L (1X 10) per well ⁵ cells/well), bEnd.3cells cell suspension was added to the upper chamber while 600. Mu.L of complete medium was added to the lower chamber, and the culture was continued in a cell culture vessel. Until bEnd.3cell monolayers exceeded 300 Ω cm2, suggesting that modeling was successful and in vitro BBB model experiments were possible. (alternatively, 200. Mu.l of culture medium was added to the upper chamber and the liquid level did not drop after 3 hours, indicating success of modeling)

8. Cell inoculation: n2a cells at 2X 10 ⁵ The density of individual cells/wells was seeded in the lower chamber. And (3) paving cells in a proper state into 24 pore plates uniformly, keeping the density of each pore at 50% -60%, and ensuring that the cells are not overlapped and not agglomerated. Incubate (overnight) to cell attachment.

9. Administration: grouping: (1) PBS-treated group (PBS + fresh medium). (2) FAM labeled PROTAC treatment group (FAM-PROTACs + fresh medium) (3 time gradients). (3) FAM-labeled PROTACs-NPs treatment group (FAM-PROTACs-NPs+fresh medium+N2a cells) (3 time gradients). Each group was provided with 3 duplicate wells, a total of 27 duplicate wells. And (3) drug treatment: FAM-labeled PROTACs-NPs (20. Mu.M), FAM-labeled PROTAC (20. Mu.M) and equal volumes of PBS were added to the upper chambers of each group of Transwell plates, respectively, and incubation was continued for a certain period of time (3, 6, 9 h).

10. Detecting the fluorescence intensity of the lower culture medium: after incubation for a certain period of time, the Transwell plate was removed and the fluorescence intensity of free FAM-PROTAC or FAM-PROTACs-NPs in the lower chamber was measured using a fluorescence spectrophotometer (Hitachi, F-4700) or a microplate reader with excitation wavelength of 518 nm.

11. Fixing and dyeing: the lower chamber was aspirated to discard the medium. 400 μl PBS was added to each well and washed twice at about 10r/min on a shaker for 5min each. 400 μl of 4% paraformaldehyde fixative was added to fix for 20min. The cells were washed twice again with PBS under the same conditions as above.

12. Shooting: and (3) taking the DAPI staining reagent, dripping 60 mu l of the DAPI staining reagent on a glass slide per hole, and buckling the cell climbing sheet on the DAPI reagent to ensure that the cell surface is contacted with the staining reagent. Each slide can hold 2 cell slide. Shooting under a fluorescence inverted microscope after preparation. The cellular nanocomposite uptake was monitored at different time points by fluorescent microscopy.

In this example, the blood-brain barrier transport capacity of PROTACs-NPs was studied in vitro using a conventional 2D model. Briefly, bEnd.3 cells were seeded on transwell's ECM precoat membrane to mimic the BBB, while N2a cells were seeded into the basal lumen to create an extraluminal (brain-facing) environment. FAM is a fluorescent dye coupled to a PROTAC molecule to quantitatively analyze the amount of drug across the blood brain barrier by detecting the fluorescent signal from the medium in the bottom chamber. As shown in FIG. 9, the fluorescence signal of the FAM-PROTACs-NPs treated group was significantly stronger than that of the FAM-PROTACs treated group, indicating that NPs promoted the BBB transport efficiency of PROTAC molecules. Importantly, fluorescent signals were also detected in N2a cells, indicating that NPs successfully delivered PROTAC to neuronal cells. Furthermore, the FAM-PROTACs-NPs treated group showed a greater advantage in accumulation of FAM-PROTACs in N2a cells compared to the FAM-PROTAC control group (fig. 10), indicating that NPs promoted uptake of PROTACs in neuronal cells.

WesternBlot validation ProTACs-NPs nanoformulations induce clearance of tau protein in N2a cells overexpressing tau

1. Detection of K5-EGFP-Tau plasmid transfected cells and transfection effect

(1) Plasmid transfected cells were performed when the seeded cells in 6-well plates grew to 70-90% confluency.

(2) Dilution with Opti-MEM MediumThe reagent (2 tubes) was thoroughly mixed.

(3) The plasmid DNA was diluted with Opti-MEM medium to prepare a DNA premix, and then P3000 was added ^TM The reagent is fully and evenly mixed.

(4) Diluted in each tubeDiluted DNA was added to the reagent (1:1 ratio).

(5) Incubate for 5 minutes at room temperature.

(6) The DNA-liposome complex was added drop-wise to the 6-well plate medium with cells spread, and the cells were incubated in a constant temperature incubator overnight.

(7) The next day, the expression of green fluorescent protein in the cells was observed under a fluorescent microscope.

2. Cell seeding

N2a cells in the logarithmic growth phase were taken, medium was discarded and washed 1-2 times with 2ml PBS, digested with 1ml pancreatin for 1min, stopped, centrifuged, and cells were resuspended. After counting by a cytometer, the cell density was adjusted to 1×10 ⁶ And each ml. N2a cells were then plated into 6-well plates, 1ml of cell suspension was added to each well, 1.5ml of fresh medium was added, and the cells were thoroughly shaken and placed in an incubator (37 ℃,5% CO 2) for overnight culture. Plasmid transfection of N2a cells was performed the next day.

3. Experimental grouping and drug configuration

The experiments were divided into 5 groups: (1) a blank group (N2 a cells after plasmid transfection+complete medium), (2) a PROTAC drug group 2 group (N2 a cells after plasmid transfection+complete medium containing PROTAC drug at 10. Mu.M and 20. Mu.M in terms of PROTAC), (3) a PROTACs-NPs nanoformulation group 2 group (N2 a cells after plasmid transfection+complete medium containing PROTACs-NPs nanoformulation at 10. Mu.M or 20. Mu.M in terms of PROTAC). The above groups are all provided with 3 complex holes. The previously prepared ProTACs-NPs nanofabricated or PROTAC solution was taken and stored in a refrigerator at 4℃and 0.5ml each, 1.5ml of complete medium was added, and after thorough mixing, the medium was added to dilute according to concentration gradient to give final concentrations of 10. Mu.M and 20. Mu.M.

4. Drug treatment

After overnight cell culture, cells were observed to adhere to and grow well by a microscope, old medium was aspirated, washed 2 times with PBS, and 2.5ml of the medium corresponding to each group was sequentially added to each well. The cells were placed in an incubator for further 24 hours, and then removed and cellular proteins were extracted.

5. Extraction of intracellular proteins

The 6-well plate was removed, old medium was aspirated, washed 2 times with pre-chilled PBS, and the 6-well plate was placed flat on ice. Adding 100 μl of cell lysate (containing 1% PMSF) into each well, shaking to uniformly cover the bottom surface of the whole well, and placing on ice for full lysis for 30min; the cell lysates were then carefully scraped to one side of the 6-well plate with a cell scraper, transferred to different EP tubes with a pipette, centrifuged for 15min (4 ℃,12000 rpm) and after careful pipetting of the supernatant into clean 1.5ml EP (avoiding aspiration of the lower sediment) and kept in a-20 ℃ refrigerator for further use.

6. BCA kit protein quantification

(1) Preparation of BCA working fluid

The total volume of BCA working fluid required for the experiment was calculated in advance using the following formula:

vtotal= (number of protein standards+number of samples to be tested) × (volume of BCA working fluid required for each sample) ×number of wells

In the experiment, 5 groups of samples to be detected are used, 8 concentration groups of protein standard substances are used, more than 3 compound holes are formed, 180 mu L of BCA working solution is needed for each hole of a 96-hole plate, and 7020 mu L of BCA working solution is needed according to the formula. And uniformly mixing the reagent A and the reagent B of the BCA kit according to the volume ratio of 50:1, and preparing 7500 mu L of working solution altogether.

(2) Preparation of standards

The BCA standard was aspirated at 5mg/mL and diluted to different concentration gradients with PBS, respectively: 0. 25, 50, 100, 200, 300, 400, 500 μg/mL.

(3) Preparation of the sample to be tested

mu.L of the experimental test sample was aspirated and diluted 10-fold with PBS.

(4) BCA assay

20 μl of the diluted standard and the sample to be tested were aspirated separately and added to each well of the 96-well plate, each group being 3 multiplex wells. 180. Mu.l BCA working fluid was then added to each well and gently shaken for 30s. Then placing the 96-well plate in an incubator at 37 ℃ for incubation for 0.5-1h, measuring the OD value of each well at 562nm wavelength by using an enzyme-labeled instrument, making a standard curve, and calculating the protein concentration of each sample to be detected.

(5) Protein denaturation

According to the protein loading amount of 40 mug of each hole during electrophoresis, the volume of the required protein solution is converted, and 1/4 volume of 5 XLoadingBuffer buffer solution is added to calculate the loading volume. Centrifuging with a centrifuge after fully mixing, boiling in water bath at 100deg.C for 10min for denaturation, centrifuging again, and storing in a refrigerator at-20deg.C.

7. SDS-PAGE electrophoresis

(1) Glue making

The protein electrophoresis gel used in the experiment is a 12.5% system. Before glue is dispensed, the short glass plate and the long glass plate are cleaned and aligned, carefully placed in a glue making frame and clamped, and vertically clamped on a glue filling frame. The clear water can be fully filled between the two glass plates to check whether the liquid leakage exists, if the liquid leakage exists, the installation is needed again, and if the liquid leakage does not exist, the clear water can be carefully poured out. And (3) avoiding the glass plate from shifting, and filling glue after glue preparation is completed. And (3) a glue preparation flow: (taking a piece of glue of 0.75/1.0/1.5mm as an example)

1) Mixing the same volume of the separation gel buffer solution and the separation gel solution uniformly, namely taking 2.0/2.7/4.0mL of each of the two solutions.

2) And (3) adding 40/60/80 mu L of modified ammonium persulfate solution (which is solidified too quickly and can reduce the use amount of ammonium persulfate by half) into the mixed solution in the step (1), and fully and uniformly mixing.

3) And (3) injecting the solution in the step (2) into the glued glass plate. Note that the gel mold was filled with the concentrated gel within 2 minutes after the addition of the separator gel, and the gel was slowly filled to prevent the mixture of the concentrated gel and the separator gel. If the individual feels difficult to prepare, the method can be changed into the method that the concentrated glue is prepared after the self-sealing with isopropanol.

4) Preparing concentrated glue: mixing the concentrated gel buffer solution and the concentrated gel solution uniformly, namely taking 0.5/0.75/1.0mL of each of the two solutions, adding 10/15/20 mu L of modified ammonium persulfate solution, and fully mixing uniformly.

5) Injecting into the glued glass plate, and inserting comb teeth (the glue is not inserted hard and gently when being inserted into the comb).

6) After the concentrated gel is solidified for 15min, the comb teeth are pulled out for electrophoresis. And (3) injection: please use freshly prepared running buffer as much as possible.

(2) Loading sample

And (3) preparing an electrophoresis liquid: deionized water 900ml, SDS-PAGE electrophoresis (Tris-Gly, 10X) 100ml, and magnetic stirring to dissolve it well. And taking the glue making frame off the glue filling frame, taking out the glass plate, putting the glass plate into an electrophoresis tank (taking care of making the sample adding tank upward, the long glass plate outward and the short glass plate inward), and adding electrophoresis liquid to make the liquid level higher than the upper edge of the short glass plate. The pre-treated protein sample and the Leader were added to each well in sequence in calculated volumes using a pipette, taking care to avoid spillage and bubble generation.

(3) Electrophoresis

The voltage is set at 80V at the beginning of electrophoresis, after the molecular weight proteins in the Leader are clearly dispersed and distributed on the gel, the voltage can be readjusted to 130V, and the electrophoresis is stopped when the first strip of the indicator reaches 1-2cm away from the lower edge of the long glass plate.

8. Transfer film

(1) Preparing a transfer buffer solution: 100mL of 10 Xelectrophoresis transfer buffer solution is taken, 200mL of absolute methanol is added for uniform mixing, distilled water or deionized water is added for constant volume to 1L, and the mixture is used after uniform mixing. After being sufficiently dissolved by magnetic stirring, the mixture was poured into a tray. The glass plate was carefully removed from the electrophoresis tank, gently pried off with a plastic spatula and the concentrated glue was peeled off, the excess of the separation glue was carefully peeled off, and the remaining separation glue was immersed in the transfer buffer.

(2) 1 NC membrane and 6 filter papers are prepared, the NC membrane is cut to a size which can completely cover the residual separating gel, and the NC membrane is put into a transfer membrane buffer solution for soaking. Taking out and opening the gel support transfer clamp, putting into transfer buffer solution, keeping the black surface at the lower part and the level, sequentially filling 1 sponge pad and 3 layers of filter paper, and repeatedly rolling by a roller to squeeze out bubbles. And taking out the separating glue carefully, lightly placing the separating glue on filter paper, covering the cut NC film on the upper layer of the separating glue, fully covering the separating glue, then sequentially filling 3 pieces of filter paper and 1 piece of foam cushion on the upper layer of the film, continuously repeatedly rolling by using a roller, and closing a transfer printing clamp after no bubbles are generated. ( Note that: all the above operations are carried out by immersing in a transfer buffer. )

(3) The transfer nip is placed in the electrical transfer module with the white face of the transfer nip against the red face of the electrical transfer module and the black face of the transfer nip against the black face of the electrical transfer module. And then the electric transfer printing module is placed on one side of a buffer liquid tank filled with transfer film liquid, and meanwhile, an ice bag is placed on the other side of the buffer liquid tank.

(4) The protein in the separating glue can be transferred to the NC film by 250mA for 90min and can be properly prolonged until the protein in the separating glue is completely transferred to the NC film.

9. Immune response

(1) After the transfer of the membrane is completed, the membrane is carefully taken out, and is dyed with 1X ponceau dye liquor and placed on a shaker for 5min, and after all the strips develop, the strips with the required molecular weight are carefully sheared. The membrane was then washed 3 times with TBST on a shaker until ponceau was completely washed clean. (TBST is isotonic buffer salt solution containing 0.5 per mill Tween 20.) (2) placing the clean membrane in an incubation box containing a proper amount of sealing solution, sealing on a shaker at room temperature for about 1h, and then washing with PBST for 3 times each for 5-10min. ( And (3) injection: the sealing liquid is PBS solution containing 5% skimmed milk powder, and is thoroughly mixed by vortex vibration in advance. )

(2) Tau, p-tau and beta-actin primary antibodies are diluted by using a Western primary antibody diluent according to the volume ratio of 1:1000, and the diluted primary antibody solution and the corresponding protein strips are incubated and placed in a refrigerator at 4 ℃ for overnight. The next day the incubation was performed 3 times with TBST on a shaker for 5-10min each time.

(3) Diluting the secondary antibody by using a Western secondary antibody diluent according to the volume ratio of 1:1000, incubating the diluted secondary antibody solution and the corresponding protein strips for 1-2h at room temperature in a dark place, and washing the secondary antibody solution on a shaking table for 3 times in a dark place by using TBST (Tunnel boring test) for 5-10min each time.

10. Film sweeping

After NC film scanning using Odyssey dual-color infrared laser imaging system, data analysis of gray values was performed on the bar graph using ImageJ software.

In this example, the efficacy of PROTACs-NPs in inducing tau protein clearance in GFP-tau overexpressing N2a cells was examined directly. Cells were incubated with PROTAC or PROTACs-NPs for 24h prior to detection of tau and p-tau protein by WesternBlot assay. As shown in FIG. 11, TAU-PROTAC molecules targeting TAU proteins were found to down-regulate p-TAU proteins in addition to TAU proteins by western blot experiments. As can be seen, current TAU-PROTAC molecules lack high selectivity (and are not effective in distinguishing between TAU and p-TAU) and bind and function with p-TAU protein in addition to TAU protein. Analysis of the results of the western blot experiments demonstrated that PROTACs-NPs induced more tau protein and p-tau protein downregulation compared to PROTAC treatment, and that this downregulation showed a concentration-dependent pattern.

2.3. Co-immunoprecipitation

After 24h of drug treatment, the cells were collected in IP buffer. Co-IP analysis was performed using anti-Keap 1 antibody (10503-2-AP) conjugated to protein A/GPlus-agarose beads. Non-specific binding of target primary antibodies was assessed using normal rabbit IgG. Western blotting was performed with anti-Tau antibodies. DyLightTM 800-labeled anti-rabbit immunoglobulin G (IgG) was used as secondary antibody.

In this example, to investigate whether intracellular Keap1 protein interacts with tau in a protas-NPs dependent manner, co-IP analysis was performed in N2a cells. Cells were incubated with two concentrations of PROTACs-NPs (10, 20. Mu.M) for 24h and immunoprecipitated. As shown in FIG. 12, the results indicate that tau can co-immunoprecipitate with Keap 1. The fact that the PROTACs-NPs nano-particles can penetrate cell membranes and release PROTAC molecules, and the PROTAC molecules can be stably combined with Keap1 and target protein tau in vitro proves that the PROTACs-NPs can be effective tools for further research.

2.4. Flow cytometry verifies that PROTACs-NPs nano-preparation induces clearance of tau protein in N2a cells

1. Cell seeding

N2a cells in the logarithmic growth phase were taken, the medium was discarded and washed 1-2 times with 2ml PBS, 1ml of the digestion was performed with pancreatin, 1-2ml of the medium was stopped, centrifuged (1000 rpm,5 min), and N2a cells were resuspended in 2ml of the medium and gently blown to homogeneity. After counting by a cytometer, the cell density was adjusted to 1×10 ⁶ The cell suspension was plated at 1 ml/well into 6-well plates, thoroughly shaken and placed in a cell incubator for incubation. Plasmid transfection of N2a cells was performed the next day.

2. Experimental grouping and drug configuration

The experiments were divided into 5 groups: (1) blank (transfected cells+complete medium), (2) PROTAC drug group 2 (transfected cells+complete medium containing 10. Mu.M and 20. Mu.M of PROTAC based on PROTAC), (3) PROTACs-NPs nanoformulation group 2 (transfected cells+complete medium containing 10. Mu.M or 20. Mu.M of PROTACs-NPs based on PROTAC). The above groups are all provided with 3 complex holes. The preparation method comprises adding the PROTACs-NPs nanometer preparation or PROTAC drug prepared previously and stored in a refrigerator at 4deg.C into complete culture medium to obtain the above drugs with concentrations of 10 μm and 20 μm respectively.

3. Drug treatment

After overnight cell culture, cells were observed to adhere to and grow well by a microscope, old medium was aspirated, washed 2 times with PBS, and 2.5ml of the medium corresponding to each group was sequentially added to each well. The cells were placed in an incubator for further 24 hours, and then removed and cellular proteins were extracted.

4. Preparing a reagent:

first, a streaming wash buffer (Phosphatite-bufferedsaline (PBS), 2% fetalcalfservum, 0.1% sodium azide) and DAPI (4', 6-diamidino-2-phenylindole) were prepared. And (3) storing liquid: 70% alcohol is dissolved, the concentration is 1mg/ml, after being prepared, the mixture is wrapped by tinfoil, and the mixture can be preserved for a long time at 4 ℃ in dark place. Flow type dyeing working solution: and (3) using PBS to obtain the working solution by using 1mg/ml DAPI storage solution according to a ratio of 1:1000. For example: 1ul of 1mg/mL DAPI stock solution is added into 1mL of PBS to obtain the DAPI working solution.

5. Grouping and drug treatment:

grouping: (1) a blank control group (pbs+fresh medium+n2a cells) was included; (2) FAM-PROTACs treatment group (FAM-PROTACs + fresh medium + N2a cells); (3) FAM-PROTACs-NPs treated group (FAM-PROTACs-NPs+fresh medium+N2a cells). 0.5ml of each of the FAM-PROTACs and FAM-PROTACs-NPs formulations previously prepared and stored in a refrigerator at 4℃were transferred to separate 15ml centrifuge tubes and diluted to 20. Mu.M in fresh medium in proportion.

N2a cells were plated overnight in 12-well plates, 6 groups (4 different treatment times) were set, 3 duplicate wells each. After incubation for a period of time (6, 12, 24, 36 h) with different drugs for the next time, the supernatant was aspirated, and after washing with 500ul PBS, PBS was discarded.

6. And (3) detection:

(1) After digestion with pancreatin, the cells were blown down after neutralization with serum-containing medium and added to the flow tube.

(2) Centrifuge for 5 min (1500 g) and remove supernatant.

(3) 1mL of the flow-through washing buffer was added, washed by vortexing, and centrifuged for 5 minutes (1500 g), and the supernatant was removed.

(4) 1mL of the flow-through washing buffer was added, washed by vortexing, and centrifuged for 5 minutes (1500 g), and the supernatant was removed.

(5) 100ul of DAPI working solution was added to each tube to dilute the resuspended cells. Incubate on ice until on-press flow, which detects fluorescence intensity of intracellular FAM using flow cytometry.

^* Statistical treatment using graphpapprism 8 as statistical chart, experimental resultsAnd the statistical figures are all expressed in (mean±sem). If the two groups of measurement data obey normal distribution and variance uniformity, adopting two groups of independent sample t-test, and if the two groups of measurement data do not obey normal distribution or variance uniformity, adopting non-parameter test. Two-by-two comparisons between sets of data used one-way ANOVA analysis of variance. Double-sided p<0.05 was considered to have statistical differences.

In this example, to further examine the relationship between the clearance of PROTACs-NPs and PROTACs to intracellular tau protein and the time of drug action, quantitative detection was performed by flow cytometry. N2a cells are transfected by K5-EGFP-Tau plasmid, and the expression quantity of Tau is marked by green fluorescent protein GFP, so that the effect of drug-induced Tau protein clearance is verified. As a result, the inhibition rate of the intracellular green fluorescent protein by the PROTACs-NPs group was significantly higher than that of the PROTAC group, indicating that the PROTACs-NPs significantly enhanced the effect of PROTAC on inducing N2a intracellular tau protein clearance, and showed a significant time-dependent tau clearance effect, as shown in fig. 13.

Example 3 in vivo targeting and tissue distribution of ProTACs-NPs nanoformulations

^* The animals used in this example were SPF grade male C57BL/6 mice of 2 months of age; purchased from Shanghai university laboratory animal center. Animal experiments strictly follow relevant regulations of animal ethics.

3.1. Preparation of fluorescent dye DiR-loaded nanoparticles (DiR-NPs) and DiR-NPs distribution in mice

1. Preparation of fluorescent dye DiR-loaded nanoparticles (DiR-NPs)

The weight ratio is 10:1 (weight ratio of NT1-O16B to PBA-Q76-O16B: 3:7) and DiR were dissolved in 100% ethanol. Then 100. Mu.l of the solution was added dropwise to 300. Mu.l of sodium acetate buffer (25 mM, pH 5.2) and left to stand for 15min after brief vortexing. Then transferred to a dialysis bag and dialyzed with di h2O (deionized water) for 12 hours (1 change of fluid every 6 hours), transferred to a centrifuge tube and stored at 4 ℃ to obtain DiR-loaded nanoparticle NPs (DiR-NPs).

As a control, 200. Mu.g of DiR was dissolved in a small amount of DMSO, and then added dropwise to a PBS solution, followed by shaking to complete the preparation of the DiR-DMSO-PBS solution.

2. Distribution of DiR-NPs in mice

(1) Experimental grouping:

9 male C57BL/6 mice with the same weight are randomly divided into three groups: (1) PBS group (tail vein injection PBS); (2) DiR-DMSO-PBS group (tail vein DiR-DMSO-PBS); (3) DiR-NPs group (tail vein DiR-NPs). Wherein each of the DiR-DMSO-PBS group and the DiR-NPs group ensured that the DiR dose was 50. Mu.g.

(2) Administration of drugs

PBS, diR-DMSO-PBS, diR-NPs drugs were injected into the C57BL/6 mice of each group via tail vein, respectively. After 2 hours, the mice were anesthetized and perfused with saline. The brains and other vital organs (heart, liver, spleen and kidney) of the mice were then dissected and collected.

(3) Photographing and analysis

The isolated brain and other organs are placed in a small animal imager for shooting, so that the distribution of the medicine in the brain and other organs can be observed by visual fluorescent signals.

In this example, drugs such as DiR-DMSO-PBS and DiR-NPs were injected into C57BL/6 mice via tail vein, and after 2 hours, brains and other important organs (heart, liver, spleen and kidney) of the mice were dissected and collected and photographed in a small animal imager. The results are shown in fig. 14, which shows that: compared with the DiR-DMSO-PBS (marked as DiR), the DiR-NPs group has obviously enhanced fluorescence in each organ; in addition to the highest fluorescence intensity in the liver, there is also a stronger fluorescence in the brain. To exclude the photographing and analysis of fluorescence in the brain by fluorescence in the liver, the brains of two groups of mice were compared separately, as shown in fig. 15, and the results showed that: the significantly enhanced fluorescence in the brain of the DiR-NPs group compared to the DiR-DMSO-PBS group suggests that the fluorochromes DiR in the DiR-NPs group effectively penetrate the blood brain barrier and aggregate in the brain, increasing the BBB penetration efficiency by about 10-fold.

3.2 preparation of brain frozen section and shooting imaging analysis

1. An aluminum foil paper with the size of 3cm is taken, and one drop of embedding glue is dripped at the right center.

2. Placing the cut tissue block on embedding glue, and then dripping a drop of embedding glue on the tissue block to ensure that the tissue block is completely in the embedding glue.

3. Lightly wrapping the aluminum foil paper to ensure that the shape and the size of the tissue block are not destroyed.

4. A container is taken and a proper amount of liquid nitrogen is poured.

5. The wrapped tissue pieces were placed in a cup with isopentane (ensuring complete immersion of the tissue pieces), and then the cup was gently shaken in liquid nitrogen until the tissue pieces were completely frozen (approximately 1-2 min).

6. Taking out the tissue block, and storing in liquid nitrogen or-80% refrigerator.

7. The temperature of the slicing machine is adjusted to-20 ℃, and the slicing thickness is 4-8 microns. (sometimes with differences in demand)

8. Placing the quick-frozen tissue in a constant cooling box for balancing the temperature for 10min, and continuously slicing after finishing.

9. The sheet was attached to a slide coated with dicing adhesive. When the patch is carried out, the glass slide can be naturally stuck only by lightly leaning on the tissue slice due to the temperature difference, and the patch cannot be forced too hard so as to avoid the artificial tissue extrusion.

10. Taking out the slide, placing in a clean place, airing for 5min, and fixing for 30min by using acetone at the temperature of-20 ℃.

11. DAPI+mount was added to the slide, and the slide was stained.

12. After removal, confocal observations can be used, or immunohistochemical staining can be performed immediately or the sections can be stored at-70℃for later use.

To further demonstrate that Nanocarriers (NPs) can effectively deliver the PROTAC molecules into the brain to achieve the targeted delivery effect, in this example, cy5 fluorescent dye was used to label the PROTAC molecules, and Cy 5-PROTAC-NPs, cy5-PROTAC and PBS (as control groups) were injected into 2 month old male C57BL/6 mice, 3 mice per group, by tail vein administration, respectively. After 2 hours, the mice were anesthetized and perfused, frozen sections of the mice brains were prepared, and the fluorescence distribution in the brains of each group of mice was observed by confocal microscopy. As shown in FIG. 16, fluorescence in the brains of mice in the Cy5-PROTACs-NPs group was more pronounced, suggesting that PROTAC molecules can effectively penetrate the blood brain barrier into the brains of mice under the action of Nanocarriers (NPs) to function.

EXAMPLE 4 in vivo anti-AD Activity of ProTACs-NPs nanoformulations

^* The animals used in this example were 30+ -2 g SPF grade male APP/PS1/tau tri-transgenic mice; beijing Fukang biotechnology Co., ltd. Animal experiments were performed following the ethical guidelines of animals.

4.1. Animal model creation, grouping, administration and animal behavioural assessment (Morris water maze)

1. Animal model building and grouping process

Building and grouping animal models: genotyping was performed on purchased 3 month old tri-transgenic AD model mice to confirm that the genotyping of the mice met the experimental requirements. All protocols have been approved by the animal care and use committee of the experimental animal center at naval university. Mice were arranged at 12:12 hours day and night; food and water are available at will. After 24 identified three-transgenic AD model mice were bred to 12 months of age, the animal experiment was started. Mice were randomly divided into four groups: (1) PBS treatment group; (2) NPs treated group; (3) a PROTAC treatment group; (4) the PROTACs-NPs treatment group (n=6).

Animal administration: mice in the PROTAC-treated group and the PROTACs-NPs-treated group were treated by tail vein injection at a dose of 15mgPROTAC per kg body weight (in terms of PROTAC molecules), respectively, while mice in the PBS-treated group and the NPs-treated group were treated by tail vein injection of equal volumes/amounts of NPs. Injections were given every 3 days for a total of 10 times. After the treatment is finished, the following behavioural assessment experiment can be performed.

2. Animal behavioral assessment (Morris water maze)

The Morris water maze experiment system is composed of a water maze device, an automatic image acquisition system and a software analysis system. The water maze device mainly comprises a water pool for containing water and a round platform with movable position and adjustable height. The automatic acquisition and software analysis system of the water maze image acquires the swimming image (analog signal) of the mouse through the camera, then the swimming image is input into the image acquisition card in the computer for analog/digital conversion, the swimming analog image of the mouse is converted into a digital image and stored in the hard disk, and the digital image is subjected to image analysis to obtain related data results for evaluating the spatial memory and learning ability of the mouse.

(1) Adjusting water temperature to 25+ -1deg.C, adding appropriate amount of black ink, stirring, placing the platform in Southeast (SE) direction of water tank, and covering black cloth on the surface of the platform to ensure that the platform is not seen by mice, wherein the height of the platform is 2cm below water surface. Note that: the water temperature was checked prior to the experiment to ensure that the pool was clean and tested at fixed times per day (8:00-12:00).

(2) The periphery of the pool is shielded by a curtain, so that external shadows are prevented from being collected by software and are confused with rat shadows, black square, triangle, round and star paper cuts are respectively hung in the east, south, west and north directions of the pool wall, and because rodents are sensitive to black, the rodents are high above the sight line of the mice, and the rodents serve as space references when the mice search targets.

(3) The mice were gently placed in water facing the pool wall. Four experiments per day were performed on each mouse, and the mice were randomly placed from 2,3,8,7 (which is pseudo-random in nature, and four positions per day were used, but the order could be random), and a schematic of the pseudo-random protocol is shown in fig. 17. Putting the mice into water from the beginning of the water-entering position towards the pool wall at will, setting the swimming time to be 60s to find a hidden platform, if the platform is found successfully, recording the time for finding the hidden platform, namely the escape latency period, and enabling the mice to stay on the platform for 15s; if no platform was found within 60s, the mice were guided to rest on the platform for 15s. Note that: the operation is gentle, and the stress reaction is avoided.

(4) The mice are dried by towel or heated by a 150W incandescent lamp for 5-10min to keep warm and then put back into the squirrel cage. One direction was one round, each mouse trained 4 rounds per day for 5 consecutive days, each day in different direction sequences (see figure 17). Note that: in most studies, the above degree of training has been sufficient to distinguish between normal and impaired learning mice. However, if many mice fail to find a hidden platform within 60s at Day5, more days of training are added.

(5) After the end of the training period, day 6 was the exploration period, the platform was removed, the mice were placed in water from the opposite side of the platform quadrant (i.e., position 1) toward the pool wall, the time it entered the platform quadrant within 60s was recorded and analyzed.

In this example, four groups were set by experiment: (1) PBS treatment group; (2) NPs treated group; (3) a PROTAC treatment group; (4) TAU-PROTACs-NPs treatment group (vector of the invention). Mice were dosed by tail vein injection and their cognitive behavioral functions were evaluated by Morris water maze after the treatment period was over, as shown in FIG. 18: the 12 month old tri-transgenic 3xTg mice were significantly improved in cognitive function through the water maze after treatment with TAU-PROTACs-NPs (fig. 18A), relative to other groups (fig. 18B), e.g., latency time measured in the hidden plateau for TAU-PROTACs-NPs treated group was significantly shorter than in other groups (fig. 18C), number of plateau-free crossings significantly higher than in other groups (fig. 18D), and retention time measured in the target quadrant was significantly higher in the no plateau than in other groups (fig. 18E).

4.2 brain section preparation, fluorescent staining and analysis

1. And (3) pouring and taking brain:

(1) 4% paraformaldehyde fixing solution: dissolving PBS powder in 2L double distilled water to obtain 1 XPBS, weighing 80g paraformaldehyde powder, pouring into a conical flask containing 2LPBS, sealing, heating to 60-65deg.C on a magnetic stirrer, continuously heating and stirring until the milky suspension becomes clear and transparent, dripping NaOH solution to adjust pH to 7.3-7.4, filtering, storing in a refrigerator at 4deg.C for use, and taking care of whole-course operation in a fume hood.

(2) After deep anesthesia of the mice with 1.5% sodium pentobarbital, the limbs of the mice were fixed on foam plates with surgical tape, and the abdomen was fully exposed.

(3) The surgical forceps lift the subxiphoid skin, sequentially shear the skin, fascia and muscle along the costal arches on both sides, open the abdominal cavity, and clamp the abdominal aorta with the hemostatic forceps.

(4) The diaphragm is sheared at the joint of the diaphragm and the sternum handle, so that pneumothorax and pneumoconiosis are caused, the xiphoid process is clamped by the hemostatic forceps and lifted upwards, the diaphragm and the ribs are sheared to two sides, the heart is fully exposed, and the pericardium is peeled cleanly.

(5) The perfusion needle is inserted into the aorta from the apex of the heart through the left ventricle, and the needle is fixed for reperfusion. A small opening was cut at the right auricle, and 0.9% sterile physiological saline (about 150 ml/piece) at 4℃was first filled to replace blood until the fluid flowing out of the right auricle became clear and the eyeballs and lungs of the mice became white.

(6) The forecooled 4% paraformaldehyde (about 200 ml/mouse) was replaced by 4℃for perfusion, and forelimb twitching was seen immediately after the beginning of perfusion, and forelimb and neck stiffness was observed after the successful perfusion.

(7) Separating whole brain tissue by head breaking, preparing brain slice from left half brain, storing left half brain in 4% paraformaldehyde, and fixing at 4deg.C overnight; the right half brain is used for preparing brain tissue homogenate, and the right half brain is stored in a refrigerator at-80 ℃ or immediately prepared.

2. Preparation of tissue paraffin sections:

(1) After rat brain tissue was fixed overnight in 4% paraformaldehyde, the tissue was dehydrated by soaking in 30% sucrose solution for 2-3 days

(2) The brain tissue specimen is sequentially washed with flowing tap water for 30min, dehydrated with 70% ethanol for 30min, dehydrated with 80% ethanol for 30min, dehydrated with 95% ethanol for 1h×2 times, and dehydrated with 100% ethanol for 1h×2 times.

(3) The xylene was transparent 20min x 2 times.

(4) Wax dipping and embedding: immersing the tissue in paraffin wax at 58-60deg.C for 3 hr, repairing, and preserving at 4deg.C.

(5) Brain tissue section: paraffin-embedded brain tissue blocks were serially sectioned in coronal position with a paraffin microtome, 3-4 μm thick, fished (fished on anti-drop slides pre-treated with APES), and baked in an oven at 60-62 ℃ for 6h for subsequent immunofluorescent staining.

3. Immunofluorescent staining:

(1) Washing with water

After fixation, the sample was rinsed by cold 0.01mol/L PBS pH7.4, and finally rinsed with distilled water to prevent autofluorescence.

(2) Dyeing

1. Materials and reagents

(1) Fluorescent antibody, diluted to application concentration. (2) 0.01Mol/L PBS pH 7.4. (3) 9 parts of high-quality glycerol and 1 part of PBS (phosphate buffered saline) with pH7.4 are added to obtain the glycerol buffer solution. Glycerol has the effect of reducing nonspecific fluorescence. (4) Square plate with cover

2. Taking a fixed specimen by an indirect staining method (1), adding a primary antibody, and incubating for 30min at 37 ℃; (2) washing with PBS for 3×3 min; (3) adding the fluorescent-labeled secondary antibody, and incubating for 30min at 37 ℃; (4) washing with PBS for 3X 3 min; (5) H2O flushing and airing; (6) adding glycerol buffer solution, sealing, and performing microscopic examination.

3. Microscopic examination

The image observed by the fluorescence microscope is judged by two indexes, wherein one is morphological characteristic; the other is the brightness of fluorescence, and in the determination of the result, it is necessary to combine the two, and the determination is performed comprehensively. The image can also be generated after the section is scanned by using a section scanner, the corresponding part of the image is selected by using CaseViewer software, and the fluorescence intensity of the image is analyzed by using imageJ software.

4. Specimen preservation

Since the stability of the fluorescent dye and the protein molecule are both relative, the labeled protein may be denatured and dissociated under various conditions with the lapse of the storage time, losing its brightness and specificity. Thus, it is difficult to preserve the specimen, and therefore, the specimen should be observed immediately after the fluorescent staining is progressed. The method of preservation can be adopted as follows: (1) fixing the sample, preserving at low temperature, and dyeing at the same time; (2) the dyeing piece is sealed by adopting a high-quality sealing agent, such as a special fluorescent sealing agent or an alkaline high-quality pure glycerol sealing agent, and the like. The sealing agents can prevent fluorescence excitation and can be stored at low temperature after sealing; (4) photographs may be saved using photographing.

In this example, brain homogenates and protein samples were prepared from the mouse brain after drug treatment, and proteins such as total tau, p-tau, keap1 were examined in each group of brain protein surface by western immunoblotting (WesternBlot). From the experimental results as shown in fig. 19, it can be seen that total TAU and p-TAU in both hippocampal and cortical areas were significantly down-regulated after treatment with TAU-protas-NPs, and that the down-regulating effect on p-TAU protein was more pronounced than total TAU protein. Meanwhile, TAU-protas-NPs treated groups had a more pronounced therapeutic effect of downregulating total TAU and p-TAU compared to the PROTAC treated groups. The above results indicate that TAU-protas-NPs have better TAU protein clearance activity in mice and that p-TAU protein seems to be more easily cleared by the PROTAC-induced ubiquitin-proteasome system as a pathogenic protein relative to total TAU protein.

4.3. Preparation of animal brain homogenate and quantitative analysis of Westernblot

1. Preparation of brain homogenate:

(1) Extracting brain tissue protein with RIPA tissue lysate, and pre-cooling with a homogenizer.

(2) Taking a proper amount of tissue blocks, flushing the tissue blocks by precooled PBS, removing residual blood and impurities on the surface, and separating a required region; weighing the tissue blocks, and cutting the tissue blocks into small fragments as much as possible so as to sufficiently homogenize the tissue blocks;

(3) Transfer of tissue to RIPA tissue lysate (1% PMSF pre-loaded)

(4) Fully homogenizing (70 Hz,60s,3 times) to ensure that the lysate has no obvious tissue blocks; continuing to crack on ice for 1h;

(5) After sufficient lysis, the supernatant was centrifuged (8000 rpm,10min,4 ℃ C.);

(6) Centrifuging the supernatant again (15,000 rpm,20min, 4deg.C), and collecting the supernatant;

(7) The supernatant was taken and added with the corresponding 5XSDSLoadingbuffer, and denatured by heating (100 ℃ C., 10 min).

Remarks: the protein concentration can be measured by BCA protein quantification of the supernatants after step (6), and then the supernatants of each group can be calibrated to a uniform protein concentration with lysate.

2. Westernblot analysis

This step is referred to the Westernblot method before.

In this example, the expression level of AD-related proteins (tau and p-tau) in cells in brain-related regions of mice of the AD model can be detected by preparing the brain of the mice after drug treatment into pathological sections in combination with immunofluorescence means. The cell nucleus is green fluorescent label and the p-tau is green fluorescent label after being treated by fluorescent dye and fluorescent antibody. From the experimental results of fig. 20, it can be seen that: the p-TAU positive pathology is mainly located in the hippocampal and paraspinal cortical areas, and the disease-associated phosphorylated TAU protein (Thr 205) is significantly down-regulated in the mouse cortex and hippocampus following TAU-protas-NPs treatment. The intensity of green fluorescence was significantly reduced in the TAU-PROTACs-NPs treated group and the TAU-PROTAC treated group compared to the PBS treated group and the NPs treated group. Moreover, the TAU-PROTACs-NPs downregulation effect was more pronounced in the TAU-PROTAC treated group than in the non-nanocarrier treated group. The above results indicate that the profacs-NPs nano-preparation can effectively remove the over-expressed p-tau protein in the brain in mice, the effect of removing the p-tau protein is more obvious for brain areas (such as hippocampus and cortex) over-expressed with the p-tau protein, and the NPs can effectively deliver the profac as nano-carrier to play a larger role of removing the p-tau protein, which is consistent with the discussion results of Westernblot results of brain tissue proteins. Thus, TAU-PROTACs-NPs are thought to be effective in inducing the clearance of p-TAU protein in the brain of AD mice in vivo.

EXAMPLE 5 in vivo biocompatibility and safety of ProTACs-NPs nanoformulations

5.1. Animal weight counting and important organ sampling and slicing

The body weight of each group of mice was measured and registered every 3 days from the start of the administration treatment to the mice. After the Morris water maze experiment is finished, the mice are anesthetized with 5% chloral hydrate, and then are fixed with 4% paraformaldehyde after being perfused with physiological saline. The brain tissue (left half brain) and other important organs (heart, liver, spleen, lung, kidney) of the mice were weighed, placed in 4% poly formic acid for fixation overnight, and the weight curve of the mice during the treatment and the organ coefficients at the end of the treatment were calculated.

In this example, preliminary toxicity of PROTACs-NPs was also studied in order to further apply this nano-drug to clinic. The cytotoxicity experiment shows that the PROTACs-NPs have no obvious toxic or side effect on N2a cells below 20 mu M concentration. In the process of in-vivo experiments of animals, the toxic and side effects of medicines on organisms still need to be focused. Because the nano-drug is administrated by tail vein injection and reaches all important organs of the whole body after systemic circulation, the potential toxicity of the nano-preparation to important organs such as heart, brain, liver, spleen, lung, kidney and the like after multiple administrations is worth focusing. As shown in the experimental results of fig. 21, no significant difference was observed between the protas-NPs treated group and the other treated group in terms of weight change of mice during treatment after 10 treatments (1 administration every 3 days) at a dose of 15mg/kg (calculated as PROTAC molecule) of protas-NPs. After the water maze test, mice were sacrificed and organs such as brain, liver and kidney were taken, and the organ coefficients (organ weight/body weight×100%) of each group of mice were weighed and calculated, and as a result, as shown in fig. 22, no significant inter-group difference was observed between each treatment group.

5.2. Evaluation of liver and kidney toxicity of nanoparticles

After Morris water maze experiment is finished, the mice are anesthetized by 5% chloral hydrate, and 0.6-1ml of blood is collected by adopting an eyeball-picking blood-taking method. Standing whole blood of mice at room temperature, naturally coagulating, centrifuging at 3000r/min for 10min, collecting upper serum, placing in a 1.5ml centrifuge tube, and measuring liver and kidney function indexes such as alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), alkaline phosphatase (ALP), hematuria (Urea), uric Acid (UA) and creatinine (Cr) in the serum by using a full-automatic biochemical analyzer.

The biochemical examination is an index for assisting in diagnosing the functional state of the main system (such as digestive system, urinary system, etc.) of the organism, and the potential and difficult-to-find diseases can be found timely by the examination mode. Alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) levels are the most sensitive indicators reflecting impaired liver function. ALT is mainly present in the cytoplasm of hepatocytes, whereas AST is located in the cytoplasm and mitochondria of hepatocytes, and when hepatocytes are damaged, the permeability of the cell membrane increases, and these two enzymes overflow from the cytoplasm and enter the blood, resulting in significant elevation of ALT and AST in serum. Indicators such as Urea blood (Urea), uric Acid (UA) and creatinine (Cr) are common indicators for evaluating the renal function status, and can directly reflect the degree of injury to renal function and the quality of renal excretion function. In this example, the results of liver and kidney function examination of mice in each treatment group are shown in FIG. 23, and the results of examination of PROTACs-NPs and other treatment groups are not significantly different, indicating that PROTACs-NPs have no obvious toxic or side effects on liver and kidney function of mice.

5.3. HE staining of pathological sections of organ tissue

Fixed overnight viscera were embedded in paraffin, and serial coronal sections were made with a thickness of 6 μm. The method for making paraffin sections refers to the "brain section making" of the experimental methods section of chapter four for detailed steps. Paraffin sections are taken, xylene is dewaxed, gradient hydration is carried out on 100%, 95%, 90%, 80% and 70% ethanol, hematoxylin is added for dyeing for 10min, distilled water is used for 3 times, 1% hydrochloric acid ethanol is differentiated for a plurality of seconds, water is used for washing for 15min until the sections turn blue, excess red is counterstained for 2-5min in 0.5% eosin solution, 95% ethanol is used for washing off, dehydration is carried out on 100% ethanol, and after xylene is transparent, neutral resin sealing sheets are obtained. Morphological changes of each organ tissue were observed under an optical microscope, and photographed.

In this example, on the basis of the liver and kidney function test of mice, the heart, liver, spleen, lung, kidney and brain of mice were also subjected to pathological examination, and after hematoxylin & eosin staining (HE staining), the results are shown in fig. 24, and no obvious pathological changes were observed in each group, consistent with the results of biochemical examination. In combination with the above results, the PROTACs-NPs nano-preparation can be considered to have good in vivo biocompatibility and safety.

Claims (10)

1. A self-assembled PROTAC peptide nanocarrier, characterized in that: and loading PROTACs into neurotransmitter-like nano-carrier NPs based on calf thymus DNA intercalation technology by utilizing the negatively charged intermediate to prepare PROTAC-loaded nano-particles.

2. The self-assembled PROTAC peptide nanocarrier of claim 1, wherein:

the neurotransmitter-like nano carrier NPs are prepared from one or more mixed lipids/lipoid selected from NT1-O12B, NT1-O14B, NT1-O16B and PBA-Q76-O16B.

3. A self-assembled PROTAC peptide nanocarrier as defined in claim 2, wherein:

the mass ratio of the NT1-O16B to the PBA-Q76-O16B is 0-7:3-10.

4. A self-assembled PROTAC peptide nanocarrier as defined in claim 3, wherein:

the preparation method of neurotransmitter-like nano-carrier NPs comprises the following steps:

s1, dissolving NT1-O16B and PBA-Q76-O16B in alcohol;

s2, dropwise adding the mixed solution of the S1 into a sodium acetate solution, and then gently oscillating;

s3, dialyzing, and collecting a mixed lipid solution;

s4, adding PROTAC molecules and UPH2O into the mixed lipid solution of neurotransmitter-like nano carrier NPs, and standing at room temperature for 5-20min;

S5, adding UPH2O into calf thymus DNA, and standing at room temperature for 5-20min;

s6, dropwise adding the S5 into the S4, repeatedly blowing for a plurality of times, and standing at room temperature for 5-20min to obtain the PROTACs-NPs nano preparation.

5. Use of the self-assembled PROTAC peptide nanocarriers of any one of claims 1-4 in the manufacture of a medicament for treating or alleviating a disorder of ubiquitin-proteasome pathway dysfunction.

6. Use of the self-assembled PROTAC peptide nanocarriers of any one of claims 1-4 in the preparation of a medicament for treating alzheimer's disease or alleviating symptoms of alzheimer's disease.

7. Use of the self-assembled PROTAC peptide nanocarriers of any one of claims 1-4 in the preparation of a medicament for improving cognitive behavioral function of AD.

8. Use according to any one of claims 5-7, characterized in that:

the effective dosage of the drug, self-assembled PROTAC peptide nano-carrier is not higher than 20 mu M.

9. Use of the self-assembled PROTAC peptide nanocarriers of any of claims 1-4 in the preparation of a formulation or medicament for scavenging over-expressed pathogenic proteins p-tau.

10. An AD cell model, characterized by: the plasmid overexpressing GFP-tau was transfected into N2a cells.