Protection of Neurons Against Injury Using Neuroreceptor­ Targeting Nanoparticles

Authors: Jonathan Wang, Jared Goodman
Gainesville, FL, USA

 

Abstract

During traumatic brain injury (TBI), neurons are injured or killed by excessive amount of glutamate. Neuroprotectants can be used to rescue the neurons. Yet they are not consistently taken in by only neurons and might be relatively unstable in the circulation, resulting in a poor optimization of positive results.
In the previous year’s experiment, a device was designed to be used in the targeted delivery of the neuroprotectants to neurons. This device was an immunoliposome, which was an antibody attached to a liposome. The antibody specifically attached to a receptor on the surface of neurons alone, while the liposome was a hollow nanosphere that encapsulated a chemical and released it into the neurons. Fluorescent dyes to show that the contents of the liposomes were successfully delivered to the neurons, proving the success of the targeted delivery device.
In this year’s experiment, the hypothesis was that if drugs were used instead of fluorescent dyes, then the immunoliposomes could be used to alleviate the harmful effects of glutamate or analogue N-methyl-D-Aspartate (NMDA) (excitotoxins) by using targeted delivery to help the drugs reach the neurons.
The hypothesis was confirmed: cells treated with drug-loaded immunoliposomes showed the least amount of damage. The cells that were treated with the immunoliposomes had less breakdown of an essential protein in the cytoskeleton (alpha II-spectrin), while the cells that had not been treated with the liposomes had a much greater amount of breakdown of this protein. The results indicate that the neuron-targeting immunoliposomes are an effective method of alleviating the neuronal cell injury caused by excitotoxins.


Introduction

Every year, 270,000 people experience moderate to severe traumatic brain injury (TBI) (Penn Medicine 2006). TBI is “…an acquired injury to the brain caused by an external physical force… resulting in impairments in one or more areas, such as cognition; language; memory; attention; reasoning; abstract thinking; judgment; problem-solving; sensory, perceptual, and motor abilities; psycho­social behavior; physical functions; information processing; and speech” [United States 34 Code of Federal Regulations §300.7(c)(12)]. TBI can occur from transportation accidents, acts of violence, and sports injuries; alcohol is involved in over half of these occurrences. TBI is associated with many long-term disabilities such as Alzheimer’s and Parkinson’s disease, dementia puglistica, and post­traumatic dementia. Not all of the damage from TBI, however, occurs at the moment of injury. Secondary brain damage also occurs from the lack of oxygen to the brain after the initial injury. Another cause of secondary brain damage is the increased levels of glutamate throughout the brain. Glutamate binds to the glutamate receptors on brain cells, allowing calcium to enter the intracellular fluid. When too much calcium enters a brain cell, it leads to a number of events that eventually kill the cell. These events include reactive oxygen species, mitochondrial dysfunction, protease activation, phospholipidase activation, and calcium induced calcium release. All of these events lead to necrosis and apoptosis (programmed cell death). Certain drugs can prevent these events from occurring. They are generally called neuro-protectants. The problems with these drugs are that they are unstable while circulating in the blood (they can be excreted or metabolized). In addition, if the body is exposed to too much of these agents, there might be strong side effects, and it is difficult to give multiple drugs at the same time. This problem can be solved with the use of targeted delivery. Cancer researchers are also trying to develop targeted chemotherapy drugs to only kill the cancer cells because it would increase the effectiveness of the cancer treatment. There are several different designs for these vectors, but the basic principle involves having a nanocarrier capable of encapsulating drug molecules and coupled to an affinity ligand or antibody that targets a particular cancer cell type.

It is increasing clear that there is a promising future for the development of targeted drug delivery. Yet based on a survey of the scientific literature, targeted delivery of neuroprotecant-encapsulated nanoparticles into neurons has not been attempted.[Figure 1]

Figure 1: Cargo encapsulated by liposomes in experiment include: H2O Negati ve Control, Dextran Dye for Detection Under Microscope, Calpain Inhibitor Neuroprotectant, Caspase Inhibitor Neuroprotectant, and Combinati on Calpain/Caspase Inhibitor Neuroprotectants

Methods and Materials

Materials

Equipment used in this experiment included a Fume hood, Sets of various micropipettes (1 to 1,000 uL), Boxes of various micropipette tips, Bag of microcentrifuge tubes, Spectrophotometer, Centrifuge, Cell Incubator, Refrigerator, Freezer, Western Blot paper, Compound light microscope with a camera, Computer, 96-well plates, 12-well plates, Vacuum, Glass vacuum tips, Box of lab gloves, Lab coats, Box of parafilm, Orbital shaker, Rotatory Agitator, Vortex Genie, Heating Plate, 4-20% polyacrylamiade gel (Invitrogen), Gel Electrophoresis unit (Invitrogen), Semi-dry electrotransfer unit (Bio-­Rad), and Boiling Flasks.

Reagents used were PBS Solution, 3.7% Formalin solution, Tris-glycine electrophoresis buffer (Invitrogen), Gel transferring buffer (Invitrogen), Methanol, Tris­buffered saline with Tween-20 (TBST) Solution (Sigma), Rat Primary Cerebellar Granular Neurons, Skim Milk Powder, NMDA-R1 (extracellular loop) primary antibodies (Chemicon, #MAB363), Anti-rabbit lg­biotinylated species-donkey secondary antibody (Amersham), Anti-mouse lg-biotinylated species­sheep secondary antibody (Amersham), Strepavidin alkaline phosphatase conjugate tertiary antibody (Amersham), NBT and BCIP phosphatase substrate (KPL), SURELINKTM Chromophoric Biotin Labeling Kit (Pierce), Cell culture media, DMEM solution (Sigma), Phosphatidylcholine (Avanti Polar Lipids), Biotinylated phosphatidylethanolamine (Avanti Polar Lipids), Cholesterol (Avanti Polar Lipids), Chloroform, Dextran-Rhodamine Green (Sigma), Hoechst 33258 dye (Sigma), Liquid Nitrogen, Liposome extrusion kit, COATSOME Empty Liposomes (neutral) (NOF corp.), Texas Red conjugated Strepavidin (Rockland Inc.; #a003-09), and Dialysis kit with membrane (M.W. 20,000 cutoff).

Mechanism schematic

Identifying the correct antibody using western blot

  • Place rainbow marker, samples of control CGN into polyacrylamide gels for electrophoresis
  • Run electric current through gel at 120 volts for 90 minutes in order to separate out the different proteins by molecular weight
  • After the electrophoresis is completed, remove gel
  • Set up transfer sandwich with filter paper, gel, PVDF (Polyvinylidene difluoride) blotting membrane, and filter paper
  • Run electric current at 20 volts for 90 minutes through transfer sandwich to transfer proteins from gel to the blotting membrane
  • Take out blotting membrane and soak in solution of TBST (Tris-Buffered Saline Tween-20), milk, and primary antibody (Mouse NMDA-R Monoclonal Antibody) overnight on agitator to bind the primary antibody (NMDA-R antibody) to the proteins on the blotting paper
  • Wash with TBST on agitator to remove unbound antibodies
  • Add a solution of biotinylated secondary antibody (anti-mouse), TBST, and milk, incubate for 1 hour on agitator to bind the secondary antibody to the primary antibody
  • Wash with TBST on agitator to remove unbound antibodies
  • Add tertiary (strepavidin-alkaline phosphatase), TBST, and milk and place on agitator for 30 minutes to bind to secondary antibody
  • Wash with TBST on agitator to remove unbound antibodies
  • Develop by using BCIP (5-Bromo-4-Chloro­ 3′-Indolyphosphate p-Toluidine Salt) and NBT (Nitro-Blue Tetrazolium Chloride)
  • Observe purple colored band development

Immunocytochemistry

  • Wash CGN grown on glass cover slips with Phosphate Buffer Saline (PBS) and fixed with 4% paraformaldehyde for 10 minutes at 4°C
  • Block with a 5% Normal Goat Serum in TBST solution for 30 minutes at room temperature to prevent any non specific binding of the antibody
  • Treat one group of cells with methanol to permeablize the membrane, leaving a control group untreated with an intact cell membrane
  • Incubate both groups of cells with the primary antibody (NMDA-R antibody [1/500]) overnight
  • Wash with TBST on agitator to remove unbound antibody
  • Incubate with FITC fluorescent secondary antibody (1/1000) for 1 hour in the dark
  • Stain the nuclei of the cells with a drop of DAPI solution
  • Observe cells under fluorescent microscope

Coupling of biotin to antibodies

  • Transfer 100 µg of Mouse NMDA-R Monoclonal Antibody into a solution of biotin in DMSO
  • Add a reaction buffer to antibody and biotin solution and incubate at 37°C for 10 minutes to conjugate biotin molecules to the antibody
  • Centrifuge solution at 9,000 G’s in a filter microcentrifuge tube to remove excess biotin molecules

Creation of drug encapsulated liposomes from pre-made empty liposomes

    • Prepare contents of liposomes:
      • Calpain Inhibitor in 5% DMSO (5 mM)
      • Caspase Inhibitor in 5%DMSO (2.5 mM)
      • Dextran Green in H 2 O (2.8 mM)
      • Calpain (2.5 mM) and Caspase (1.25 mM) Combination in 5% DMSO
    • Lyophilized liposomes were rehydrated with the previous solutions to create drug­encapsulated liposomes; liposomes were also rehydrated with plain H 2 O to form a control
  • Store at 4°C

Biotinylation of drug-encapsulated liposomes with biotinylated phosphotidylethanolamine

  • Prepare a solution of biotinylated phosphotidylethanolamine in chloroform
  • Create a thin film of phosphotidylethanolamine on the bottom of the tube by evaporating the chloroform using a stream of N 2 gas
  • Incorporate the biotinylated phospholipids by adding the liposome solution to the tube of phospholipids
  • Swirl around the liposome solution to incorporate the biotinylated phospholipids into the liposomes

Conjugation of antibodies to liposomes

  • Prepare a strepavidin solution by mixing 5 mg of strepavidin with 1 mL of H 2 O
  • Add biotinylated antibody solution to the strepavidin solution
  • Provide gentle agitation for the molecule of biotin to bind to one of strepavidin’s four binding sites
  • Add biotinylated liposomes to strepavidin­antibody solution
  • Provide gentle agitation for the molecule of biotin to bind to one of strepavidin’s three remaining binding sites
  • Place the completed immunoliposome into a dialysis membrane for four hours to filter out excess molecules of Calpain Inhibitor, Caspase Inhibitor, Biotinylated phospholipids, and strepavidin that has not been incorporated into the liposome

Exposure of PC-12 cells and cerebellar granule neurons to immunoliposomes

  • Culture rat PC-12 cells or rat cerebellar granule neurons (CGN) into wells on 24-well plates with cell media
  • Differentiate PC-12 cells into neurons by exposing them to NGF (Nerve Growth Factor)
  • Maintain CGN in culture media to allow them to mature for 9-10 days in vitro
  • Expose PC-12 or CGN cells to each of the liposome conditions for 4 hours
  • Control differentiated PC-12 (no liposomes or drugs)
  • Cells exposed to neurotoxin (staurosporin STS) alone (for 16 hours) but lacking liposomes
  • Cells exposed to liposomes for 4 hours
  • Empty liposomes
  • Dextran Immunoliposomes
  • Calpain Inhibitor Immunoliposomes
  • Caspase Inhibitor Immunoliposomes
  • Calpain/Caspase Inhibitors Combined Immunoliposomes
  • After 4 hours add neurotoxin STS to the above sets to induce cell death (12 hours)

Lactate dehydrogenase (ldh) cytotoxicity assay

  • Collect 100 µL of media from each of the wells and place in microcentrifuge tube
  • Centrifuge each tube at 14,000 G’s for 3 minutes
  • Transfer 50 µL from the supernatant of each microcentrifuge tube to a well in a 96-well plate
  • Add 50 µL LDH Assay Solution to each well and let reaction proceed for 30 min in the dark
  • Measure absorption at 490 nm light wavelength in a spectrophotometer to determine cell death
  • As cells die, LDH is released, which reacts with the Assay solution to increase absorption of 490 nm wavelength of light

Spectrin breakdown analysis using western blot

  • αII-Spectrin is a protein found in the cytoskeleton of a cell. As the cell undergoes apoptosis or necrosis, αII-Spectrin begins to break down into its breakdown products fragments of 150 kDa (SBDP150), 145 kDa *SBDP145) and of 120 kDa (SBDP120). Formation of these indicated cell injury or death.
  • Samples of the cell lysate from the PC-12 cells in the previous step were collected and placed along with rainbow molecular weight marker into gels for electrophoreses
  • Western Blot technique from the above method was repeated, using the αII-Spectrin antibody as the primary antibody rather than NMDA-r antibody
  • PVDF membrane was developed using BCIP and NBT to observe purple αII­-Spectrin protein and SBDP bands

Results

Antibody-coupled immunoliposomes binding to CGN cell surface and internalization

The immunoliposomes were added to the media in the cell culture of CGN, which were then incubated at 37°C in 5% CO 2 , 95% O 2 . At the 1 and 6 hour time intervals, the CGN were removed from the incubator, and fluorescent microscopy was performed after washing the cells to remove free floating liposomes.

After 1 hour, liposomes have attached to respective receptor on the surface of the cell [Figure 2A]. Evidence of this can be seen in the fluorescent outline of the cell. The image is merged between the fluorescent image of the FITC-Dextran and the Phase contrast image of the CGN. Both pictures were taken in the same position. The fluorescence comes from the Dextran dye molecules inside the liposomes that are attached to the cell surface through the antibody binding to the receptor on the cell membrane. These were not free floating liposomes, as the media had already been removed and the cells washed with new media. This fluorescence indicates that the liposomes were able to successfully attach to the cell surface through the use of the targeting antibodies.

Figure 2A: Immunoliposomes binding to CGN cell surface and internalizati on over time

After 6 hours, liposomes have been internalized by CGN (yellow arrow), releasing their contents into the cell cytoplasm, as shown with both Dextran­green and Avidin-Texas Red fluorescence images which correspond with the locations of the cells [Figure 2B]. The contents of the liposome were released when the liposome phospholipid bilayer fused with the membrane of the cell in a process similar to endocytosis. These results indicate that not only do the liposomes bind to the cell surface, but that they are also able to release their contents into the cell.

Figure 2B: Immunoliposomes binding to CGN cell surface and internalizati on over time

Neuroprotection of differentiated PC-12 neural cells against neurotoxin

Cells except for the control and STS alone conditions were exposed to immunoliposomes for 4 hours and then, except for the control, were exposed to the neurotoxin staurosporin challenge for 12 hours [Figure 3]. Caspase and Calpain+Caspase Inhibitor immunoliposomes protected against the breakdown of cytoskeletal protein αII-Spectrin indicated by Western Blot. A greater amount of intact αII-Spectrin signifies healthier the cells, as shown by darker αII­-Spectrin band on the Western Blot and lighter Spectrin Breakdown Product bands (SBDP 150/145 kDa and 120 kDa). Cells treated with STS alone exhibited almost no intact Spectrin. Empty immunoliposomes as well as Dextran-encapsulated immunoliposomes served as negative controls. These negative controls also exhibited a minimal amount of intact Spectrin, indicating a large amount of cell death. In contrast, the amount of intact cytoskeletal protein in the Caspase and Calpain+Caspase Inhibitor conditions was comparable to healthy amounts seen in the control condition, indicating the success of the drug­-loaded immunoliposomes in preventing cytoskeletal breakdown and cell death.

 

Figure 3: Effects of various drug-loaded immunoliosomes against neuroskeletal protein áII-Spectrin proteolysis. P values obtained using a Student T-Test in comparison with the control

 

Cell death was also quantified using an LDH assay [Figure 4]. LDH release into extracellular fluid (ECF) (i.e. cell media) is proportional to the amount of cell death. LDH Assay tests were performed using the same conditions and samples obtained for the Western Blot. As before, STS, as well as the negative controls (empty liposomes and Dextran liposomes), showed significant amounts of cell death indicated by high concentrations of LDH in the cell media. Cells exposed to the Caspase and Calpain+Caspase Inhibitor immunoliposomes showed very little cell death on levels comparable to the control, showing again that the drug-loaded immunoliposome treatment was successful in preventing cell death.

Figure 4: Neuroprotective effects of various drug-loaded immunoliosomes against neurotoxic PC-12 cell death. P values obtained using a Student T-Test in comparison with the control

Neuroprotection of cerebellar granule neurons (CGN) against neurotoxin

CGN were tested in the same conditions as the PC-12 cells as stated above, using the same set of immunoliposomes as well as STS as the challenge. Exposure and incubation times also remained the same. An extra variable, the plain liposome (no attached antibody) with Calpain and Caspase inhibitors was added to demonstrate the importance of the antibody as the targeting mechanism. An LDH Assay was performed to quantify cell death. Results were similar to those obtained in the trial with the PC­12 cells, with the Caspase and Calpain+Caspase inhibitor immunoliposomes being the most effective in preventing cell death [Figure 5]. The plain liposome with the Calpain+Caspase inhibitor combination showed almost no neuroprotective qualities, and the cell death in that condition was just as high as in the STS alone condition. The difference between the Calpain+Caspase immunoliposome and the Calpain+Caspase inhibitor plain liposome was the presence of an antibody on the immunoliposomes. Taken together, the Calpain+Caspase inhibitor immunoliposome had significantly less cell death than the plain liposome, which demonstrates the effectiveness of the antibody as a targeting mechanism. This test also showed the effectiveness of the drug-loaded immunoliposomes in actual CGN neurons, not just the PC-12 cells (model for neurons) used in the previous two tests.

Figure 5: Neuroprotective effects of various drug-loaded immunoliosomes against neurotoxic challenge of cerebellar granule neurons. P values obtained using a Student T-Test in comparison with the control

Discussion

Many tests were performed to ensure the proper formation and internalization of the immunoliposomes. A Western Blot and microscopic observation were performed to display that the NMDA-R Antibody would attach to the NMDA Receptor on the neuron surface. Fluorescent dye molecules were used to obtain visual evidence of the linkage and incorporation of the separate components of the immunoliposomes. Neurons were then observed under a fluorescent microscope as the dye- loaded immunoliposomes attached to the cell surface and were internalized into the cytoplasm. These experiments displayed the ability of the immunoliposomes to be used as a targeted delivery mechanism.

The drug-loaded immunoliposome was also successful in preventing the delayed phase of cell injury in both PC-12 cells treated with NGF (used as a model for neurons) and rat primary cultured neurons (CGN). This was verified by both a Western Blot and an LDH Assay, which showed that cells treated with the drug-loaded immunoliposomes after exposure to the neurotoxin STS had significantly higher survival rates than the cells that were not treated or cells that were only treated with empty liposomes (no drug inside) or drug- loaded liposomes without the NMDA-R Antibody (lacking the targeting mechanism).

 

Conclusions

The hypothesis of this study was confirmed:

  1. Neuron-targeting immunoliposomes were successfully constructed by allowing liposomes to (i) encapsulate with Dextran-Rhodamine Green dye and (ii) conjugate to strepavidin using biotinylated phospholipid that we incorporated into the liposome’s surface, and then (iii) subsequently conjugate to the biotinylated NMDA-R
  2. These fluorescent dye-loaded immunoliposomes also successfully attached to the respective surface receptors on neurons within 1 hour and then the entire liposomes were internalized over time with their encapsulated fluorescent dyes released inside the cells thereafter (after 6 hours) [Figure 2].
  3. Neuroprotectant-loaded immunoliposomes protected PC-12 neural cells differentiated with Nerve Growth Factor against neurotoxin STS induced cytoskeleton protein αII-spectrin breakdown as measured by Western Blot [Figure 3], as well as cell death as measured LDH release [Figure 4].
  4. Neuron-targeting immunoliposomes loaded with neuroprotectants (Calpain, Caspase inhibitors) also protected rat cerebellar granule neurons against neurotoxin challenge-induced cell death as measured by LDH release [Figure 5].

About the Author

We are two 17-year old seniors from Gainesville, Florida, USA, a small college town in the heart of the state of Florida. We, combined, play three different sports for our high school while finding time to take up to five AP classes for the year. For the last three years, we have been studying nanoparticles: everything from their pharmaceutical uses to their technological uses. In our freshman year, we performed an experiment that tested the Cytotoxicity of various nanoparticles (fullerenes, carbon black, etc.) on neurons, as we were especially concerned with possible neural side effects. Because of this focus, we decided to see if there was any sort of pharmaceutical use of nanoparticles that have not been discovered yet. We continually looked, but it seemed that everything had been either discovered or tested already. But then, we thought of the current United States’ war in Iraq. We thought about how the troops were being hit by roadside bombs, suffering from head traumas that were irreversible. We thought about the people in our community who had been in car accidents, and how they, too, were suffering from irreversible head traumas. That is how we came up the idea of creating a drug that would “deliver” a drug to the specific cells in the brain that were affected by the Traumatic Brain Injury (TBI) and possibly ameliorate these terrible traumas.

Going into the Intel ISEF, we were not expecting any sort of prize. At best, we were reaching for a possible fourth place finish. We had been to the Intel ISEF before, in our freshman year, and we left empty-handed, so we knew exactly how hard it was to be successful at the competition and exactly how good everyone else in the world actually was. Little did we know that we were going to win it all! It was such an eye-opening experience winning the EUCYS competition at the ISEF in May. We had no clue that this project had people that looked at it and said, “Wow, that actually is pretty good.” We thought that we would have a repeat of our freshman year. Our experience was one to remember, and the trip we received to Denmark just thrust us into a high that we will always remember.


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