1. Nanoparticle Engineering: conjugation of peptides via E5-K5
Spencer Park
Jin Lab
8/15/08

2. Liposomes Biocompatible and biodegradable
Aqueous core entrapped by lipid bilayers
Protect encapsulated drugs from degradtion
High systemic plasma
clearance
Nanoparticle engineering has recently received substantial interest, especially in drug delivery and imaging. Liposomes have been used frequently as drug delivery vectors because of their biocompatible and biodegradable characteristics. Their aqueous core surrounded by one or more lipid bilayers has been used in protecting encapsulated drugs from degradation.
High systemic plasma clearance
Mononuclear Phagocytic System (MPS) destroy liposomes in plasma
Half-life prolonged by PEG derivatized lipids, which prevent binding of opsonins onto liposomes (Opsonin binding first step in liposome removal)
Last up to 90h in circulation (200 fold decrease in clearance)

3. Cornell Dots (C-Dots) Similar to quantum dots
Fluorescent nanoparticles (20-30 times brighter than a single dye molecule)
Resist Photobleaching
More mono-dispersed, inert, and cost effective than quantum dots
These two nanoparticles are attractive for their relatively easy surface chemistry in conjugating proteins. Molecules are attached to liposomes using the protruding amine groups, and to C-Dots using the exposed maleimides.

4. E5-K5 Acid-Base coiled coils Very strong affinity
KD ~ 60pM
Ideal system since the coils are small (38aa) and resulting non-covalent complex is stable
To increase the efficiency of peptide conjugation, acid/base coiled coils, more specifically E5-K5, will be used. E5-K5 is an acid/base coiled coil which have very strong affinity for each other. The stabilizing effect of lysine-glutamic acid pairs on the coiled coil is well documented; the association was able to be detected at a range as low as 60pM. E5-K5 coiled-coil system is ideal since the coils are small (35 amino acids) and the resulting non-covalent coiled-coil complex is very stable.

5. K5 vsalkek-vsalkek-vsalkek-vsalkek-vsalkek-ggc K - K - K - K - K -ggc
Length: 38aa
MW:
Sequence:
vsalkek-vsalkek-vsalkek-vsalkek-vsalkek-ggc
K K K K K -ggc
Cys serves as a platform for surface chemistry

6. Aims Purify K5 Conjugate K5 to nanoparticles
Bind GFP-E5 to K5-modified nanoparticles
Show multiple targeting is possible
Bind GFP and YFP (or CFP) simultaneously
Replace GFP with any protein of interest
Targeting agents (i.e. herceptin to target Her2-positive breast cancer)
In my effort of protein conjugation via acid/base coiled coils, the following are the immediate goals: (1) purifying K5, (2) conjugation of K5 to nanoparticles, and (3) binding GFP(YFP, or CFP)-E5 to K5-modified fluorescence-encapsulating nanoparticles. Successful binding of GFP-E5 along with YFP-E5 or CFP-E5 will show that multiple targeting is possible, which will in turn increase targeting specificity and allow for simultaneous targeting and imaging. Eventually, GFP will be replaced with any protein of interest, such as herceptin in targeting Her2-positive breast cancer.

7. Our Methods

8. Nanoparticle Production
Liposome
Use lipid-film method and 3:1:1 ratio of phosphatidylcholine (PC), diplmitoylphosphatidylethanolamine (DPPE), and cholesterol (Chol) (~100nm)
C-Dot provided by Uli Wiesner

9. Addressing problem of immune response
Coat liposomes with hyaluronan or heparin
HA & HP exist in our bodies and cause little immune response
Also cryoprotectors during entrapment
Cholesterol included in liposomes to prevent lipid interactions with HDL or LDL
Interaction could cause rapid release of encapsulated drug
Coat C-Dots with PEG, hyaluronan or heparin
PEG prevents opsonin binding, which is the first step in plasma clearance by the phagocytic system
One possible downside of using liposomes is their high systemic plasma clearance level; liposomes are tagged with opsonins, which are then recognized by mononuclear phagocytic system (MPS) as foreign substances and destroyed.6 To prevent this phenomenon, we will be coating the liposomes with either hyaluronan or heparin, both of which already exist in our bodies and therefore cause no unwanted immune response.7 In addition to phosphatidylcholine(PC) and dipalmitoylphosphatidylethanolamine (DPPE), cholesterol will be included in making liposomes to prevent interactions with HDL and LDL, which can cause rapid release of encapsulated drug into the plasma.6 For the same reason, I will treat the C-Dots with PEG, hyaluronan or heparin.

10. Purification
Decorated nanoparticles purified via size exclusion to remove unattached K5
Measure particle diameters and surface charges using Zetasizer
Measure number of K5 per nanoparticle to assess coupling efficiency

11. Surface Chemistry Options
PDEA & thiol coupling
Ethlyene diamine & S-SMCC
No HP or HA
Which ones maintain the ability to bind E5 after a cycle of lyophilization and rehydration?
The targeting agent will then be attached to either the sugar-coated liposomes or C-Dots via E5-K5. A several surface chemistry options will be explored to attach K5 onto the liposomes. Heparin or hyaluronan will be attached to liposomes by using the amine groups on DPPE. The first method utilizes PDEA & thiol coupling, where the carboxyl groups on either heparin or hyaluronan will be activated by NHS/EDC to allow binding of PDEA and subsequently, K5. The second method will attach ethylene diamine to the carboxyl groups of the sugar coat and use the S-SMCC method to attach K5. The last method involves having no sugar coating and attaching K5 directly onto the amine groups of DPPE via S-SMCC. In the end, we will compare the three methods to study which ones maintain the ability to bind E5 after a cycle of lyophilization and rehydration, a method that will be used for encapsulation. Since heparin and hyaluronan are cryoprotectors as well, we predict those vectors with the sugar coating will be more effective in binding E5.

12. PDEA & Thiol Coupling O COOH COON O CONH CONH PDEA NHS/EDC PDEA SS
Ligand-SH
Ligand
CONH SS

13. Ethylene diamine & S-SMCC
NH2
COOH
COON
Ethylene diamine
S-SMCC & K5 O S NH N K5
COON O O

14. Direct Addition of K5
S-SMCC & K5
NH2 O S NH N K5 O O

15. C-Dot Surface Chemistry
Use maleimides exposed on surface of C-Dots
Double bond react quickly with the thiol group of K5 to form a stable carbon-sulfur bond HN
The surface chemistry of C-Dots will be simpler, using maleimides exposed on the nanoparticles. Maleimides linked to polyethylene glycol chains are often used as linking molecules to attach proteins to surfaces. The double bond should quickly react with the thiol group found on cysteine of K5 to form a stable carbon-sulfur bond. O O

16. Addition of Targeting Agents
Control number of targeting agents attached by limiting the amount of targeting agent-E5 that are allowed to react with K5
Assay must be carried out to show E5 and K5 are functional and that conjugation is done via E5-K5
Study binding efficiency of targeting agents with and w/o E5-K5
Show E5-K5 is necessary for efficient and stable conjugation
Dynamic light scattering to measure size and zeta potential (narrow range is desired)
Once the nanoparticles are covered with K5, controlling the number of targeting agents fused onto the particles will be done by controlling the amount of targeting agent-E5 that are allowed to react with the K5. Since too much of targeting agents will increase the sensitivity and cause unwanted binding to normal level expressions of target proteins and too little will decrease the binding efficiency, controlling the number of targeting agents on nanoparticles is crucial. An assay must be carried out to show that E5 and K5 and functional and that the conjugation is done via the coiled coil only. In addition, a study of the binding efficiency of targeting agents with and without E5-K5 should be done to show that E5-K5 is necessary for efficient and stable conjugation. At this step, dynamic light scattering will be used to measure the range of size and zeta potential of the nanoparticles; a narrow range of size is desired.

17. Possible in vitro experiments
Study targeting efficiency
Transfect Her2 expressing cells with nanoparticles conjugated with herceptin and GFP
Liposome
C-Dots
Compare with control cell line (HeLa)
Eventually, in vitro experiments will be carried out to study the targeting efficiency. For example, Her2 expressing cells will be transfected with nanoparticles conjugated with herceptin and GFP. In the case of liposomes, if endocytosis takes place after binding to Her2, GFP should be observed inside the cells, while C-Dots should be coating the surface of cells. The results will be compared to a control cell line transfection, such as HeLa.

18. Possible in vitro experiments (cont.)
Competition Experiment
Incubate Her2-positive cells with varying concentrations of free herceptin and herceptin-CDOT
As concentration of free herceptin increases, GFP signal inside/ or coating the cell will decrease
Shows herceptin - Her2 targeting is efficient

19. Possible Future Explorations

20. Drug + Imaging Conjugation of both a drug with an imaging agent
Study pharmacokinetics of drugs and monitor diseased tissue during treatment
Requires no administration of a separate contrast agent
Conjugation of both a drug with an imaging agent
Study the pharmacokinetics of drugs and monitor diseased tissue during treatment
Requires no administration of a separate contrast agent

21. Membrane Disruption for Liposome Uptake
Reovirus Fusion-Associated Small Transmembrane Protein (FAST protein)
p14 FAST protein is the simplest known membrane fusion protein that is both necessary and sufficient for liposome-cell fusion, in addition to cell-cell fusion

22. Possible Uses of Liposomes
Cationic liposomes for gene delivery
Liposomes for diagnostic imaging
Encapsulation of air to be echogenic for ultrasound
Liposomal gadolinium for MRI
BBB Targeting using OX26 monoclonal Ab (cationized albumin)
Cross BBB via receptor-mediated transcytosis (transferrin receptor)
pH sensitive liposomes release content when pH decreases in endosomes after receptor-mediated endocytosis
Exploit liposomes’ rapid capture by the phagocytic system to deliver drugs to treat infections in the phagocytic system

23. Thus far… Purifying K5 K5 dissolved in 1X PBS (pH7.4)
Purified using FPLC
15% SDS PAGE (reducing & non-reducing)
Ellman’s Assay to test for presence of free thiol groups
Standard Curve using L-Cys
Thus far, I have been working on purifying the base coils (K5). K5 dissolved in 1X PBS (pH7.4) was purified using the FPLC. In order to take into account the possibility of di-sulfide bond cross-linking, we ran the 15% SDS PAGE gel under reducing and non-reducing conditions. There was not much difference between the two showing that most are present in the monomeric form. The candidates were then tested for free Cys using Ellman’s Assay. L-Cys was used to create the standard curve to calculate the concentrations of free -SH. Interestingly, our results from the Ellman’s Assay do not correlate with our FPLC data. Possibly, the K5’s with free thiol groups may be binding to the column and not being eluted. Moreover, though K5 (~4kDa) is expected to be eluted from FPLC around 15-18mL, the elutes have been coming out at least 5mL ahead of the expected range. This may be because K5 is still in aggregated form in PBS or are forming dimers. A better method of dissolving K5 in PBS, such as sonication, will be explored to address this problem.

24. K5 FPLC A1 A3 A7 A12 C3 C7 Reason for picking these samples:
A1-A3: Aggregates
A7-A12: Highest Peak even though very early
C3-C7: Expected range for K5 (4kDa)

25. 15% SDS PAGE K5 FPLC Samples
M A1 A2 A3 A7 A8 A9 A10 A11 A12 C3 C4 C5 C6 C7

26. K5 FPLC A7
A12

27. Discrepancies Ellman’s Assay (detect free thiol groups)
A7-A12 do not show heightened level of absorbance
Possible explanations
K5’s are in aggregate form and hiding thiol groups
K5’s are forming di-sulfide bonds