Nanoscale functionalities for biopharmaceutical drug delivery

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Nanoscale functionalities for biopharmaceutical
drug delivery

• Gorazd Hribar, Andrej nidaric, Marjan Bele,
  Simon Caserman, Peter Venturini, and Vladka
  Gaberc-Porekar

National Institute of Chemistry, Hajdrihova 19,
SI-1000 Ljubljana, Slovenia E-mail
gorazd.hribar_at_ki.si
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1. Protein self-assembly
Concept Designed proteins capable of forming
aggregates nanoparticles by self-assembly
(H7dN6TNF, His10-TNF).
biocompatible chelate
TNF analogue
Aim Controlled formation of His-tagged protein
aggregates and nanoparticles by polyfunctional
biocompatible chelates.
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2. Proteins on inorganic nanoparticles
Concept Nanoparticles decorated with specially
designed TNF analogues. (H7dN6TNF, His10-TNF)
Aim Preparation of protein-decorated
nanoparticles for increased immunogenicity of an
antigen, e.g., TNF.
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Model proteins Histidine-rich TNF-alpha
analogues
Preparing of histidine rich TNF-alpha
analogues. Coordinative binding of transition
metal M2 ions to histidine tags or surface
clusters of histidines.
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TNF-alpha

• Tumor necrosis factor alpha.
• Pleiotropic inflammatory cytokine.
• Number of functions. TNF causes apoptotic cell
  death, cellular proliferation, differentiation,
  inflammation, tumorigenesis and viral
  replication.
• Low levels of TNF remodeling or replacement of
  injured tissue. Immune response to bacterial, and
  certain fungal, viral, and parasitic invasions.
  Necrosis of specific tumors. Key mediary in the
  local inflammatory immune response.

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Clinical applications

• TNF-alpha antitumor activity use limited on
  local therapies due to its high toxicity.
• Anti-cancer therapy fusion of TNF with other
  molecules (monoclonal antibodies), which can
  specifically bind to tumor tissue.
• Inhibition of effects of high levels of TNF in
  diseases such as rheumatoid arthritis, Chron
  disease, psoriasis, AIDS, bacterial septic shock.
  Strategies for preventing TNF activity include
  neutralization of the cytokine via either
  anti-TNF antibodies, soluble receptors, or
  receptor fusion proteins.

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Potential use

• Upon administration of protein aggregates an
  increased immune response is anticipated.
• Enhanced formation of antibodies against
  TNF-alpha would be advantageous serving as a
  basis for developing new drugs for chronic
  diseases associated with pathogenically elevated
  TNF-alpha levels (rheumatoid arthritis, Crohn
  disease, psoriasis, etc.).
• His10-TNF and H7dN6TNF analogues especially
  interesting, since they exhibit very low in vitro
  cytotoxic activity. Upon formation of
  nanostructures, a significantly diminished number
  of accessible receptor binding sites and
  consequently even more reduced cytotoxicity is
  expected, leading to safe formation of
  anti-TNF-alpha antibodies.
• Protein nanostructures with LK-801 sustained
  release due to lower pH of tumors. Antitimor
  activity (treatment of solid tumours).

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Preparation of analogues

• Preparation of plasmids in E. coli DH5? strain.
• Production of recombinant proteins in 5l
  bioreactors in E. coli BL21(DE3) strains using
  low temperature fermentation.
• Purification of analogues using IMAC (50-100 mg).
• Characterisation of analogues
• electrophoretic
• (SDS-PAGE, IEF)
• chromatographic
• (SE-HPLC)
• spectroscopic methods
• (UV-VIS)
• Determination of
• biological activity.

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Isolation of His10-TNF analogue on IMAC

• A1 A2 buffer 25mM imidazole
• A2 20mM K-phosphate, 0,5M NaCl, pH 7,2
• B1 A2 buffer 150mM imidazole 0,1NLS
• C1 A2 buffer 500mM imidazole
• C2 A2 buffer 50mM EDTA
• Matrix Cu-IDA Vcolumn 30ml

Single step chromatographic isolation of
recombinant protein. Amount of purified protein
80 mg His10-TNF
M12 standard Mark 12 S
starting material UN unbound proteins
B1 elution with B1 buffer A3-D12
analysis of fractions C2e elution with
C2 buffer
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Isolation of LK-801 analogue on IMAC
A phosphate buffer (20mM K-phosphate,
0,2M NaCl, pH 7,1) B buffer A 100mM
imidazole, pH 7,1 Matrix Zn-IDA Vcolumn 30ml

• Single step chromatographic isolation of
• recombinant protein.
• Amount of purified protein 106 mg LK-801

LMW low molecular weight standard
S starting material UN unbound proteins
1.P 1.
peak A13 fraction A13
3.P 3. peak E1-G15
analysis of fractions
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Isolation of H7dN6TNF analogue on IMAC
A phosphate buffer (20mM K-phosphate,
0,2M NaCl, pH 7,2) B buffer A 100mM
imidazole, pH 7,2 C buffer A 50mM
EDTA Matrix Zn-IDA Vcolumn 30ml

• Single step chromatographic isolation of
• recombinant protein.
• Amount of purified protein 87 mg H7dN6TNF

LMW S UN 1.P EDTA
FR1.........LMW...................................
..FR20
LMW low molecular weight standard S
Starting material UN unbound proteins
1.P 1. peak EDTA elution
with EDTA FR 1-20
fractions with pure H7dN6TNF
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Preparation of inorganic nanoparticles

• Inorganic Zn-phosphate nanoparticles prepared by
  precipitation reaction (1h) at RT and pH around
  7.0.

Zn-phosphate nanoparticles with bound His10-TNF
and H7dN6TNF observed under electron microscope.
Particles are uniform in size between 40 - 50
nm.
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Binding on Zn-phosphate nanoparticles

• Analysis SDS-PAGE nanoparticles washed and
  elution with buffer with lower pH or with buffer
  with 0,5 M imidazole present.
• Bound BSA and TNF
• analogues are released.

LMW - low molecular weight standard UB unbound
proteins (2 His10TNF 3 LK-801, 4
H7dN6TNF) W washed particles with buffer with
pH 7.2 no release of protein B release of
TNF-alpha analogues due to addition of imidazole
(2 His10TNF 3 LK-801, 4 H7dN6TNF)
LMW low molecular weight standard pH1, pH2
elution with buffer with lower pH IMI1, IMI2
elution with buffer with 0,5M
imidazole
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• The release from inorganic nanoparticles was
  achieved under very mild conditions during 2
  weeks close to phisiological conditions (pH
  around 6,0 or citrate present in 0,5 mM
  concentrations) useful for developing
  formulations with prolonged release.
• Released TNF-alpha analogues retained their
  biological activity (measured as specific
  cytotoxicity on mouse fibroblast L929 cell line)
  binding is reversible.

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Comercially obtained silica nanoparticles
prepared by homogenization (with ultrasound),
Zn-acetate added and stirred overnight. Size
around 20 nm. TEM image.
Zn-silica nanoparticles
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Binding on Zn-SiO2 nanoparticles

• Zn-silica nanoparticles prepared 20 nm in
  diameter and bovine serum
• albumin bound on Zn2 coordinative binding.

• Lanes 1, 2 and 3 - analysis of BSA binding to
  zinc silica nanoparticles.
• Lanes 4 and 5 - binding of BSA to silica
  nanoparticles without addition of zinc.
• Bound BSA is released.

LMW (low molecular weight standards,
BioRad) Lanes 1 and 4 - unbound BSA Lanes 2 and
5 - release of BSA due to addition of
imidazole Lane 2 - washed particles with
phosphate buffer with pH 7.2 no release of BSA
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Chelators used in aggregation experiments
1,4,8,11-Tetraazacyclotetradecane- 1,4,8,11-tetraa
cetic acid - TETA
Phytic acid
Aggregates under EM. If too much zinc is added,
you can not control the aggregation. Zinc added
at the end control of pH (around 7.0).
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SEC size exclusion chromatography
Superdex 200 10/300GL Vcolumn24ml
Principle of SEC separation due to the size of
the molecule. Bigger molecules come out first.
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DLS dynamic light scattering

• Protein self-assembly
• H7dN6TNF
• H7dN6TNF Zn2
• H7dN6TNF Zn2 phytic acid
• H7dN6TNF Zn2 TETA
• The key is to control the amount of zinc ions.
• DLS can be used to determine the size
• distribution profile of small particles in
  solution.

1,4,8,11-Tetraazacyclotetradecane-
1,4,8,11-tetraacetic acid
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H7dN6TNF 1,12mM Zn2
H7dN6TNF
H7dN6TNF 2,25mM Zn2
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H7dN6TNF 1,5mM Zn2 phytic acid
H7dN6TNF 0,75mM Zn2 phytic acid
H7dN6TNF 2,25mM Zn2 phytic acid
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H7dN6TNF 1,5mM Zn2 TETA
H7dN6TNF 0,75mM Zn2 TETA
H7dN6TNF 2,25mM Zn2 TETA
H7dN6TNF 3,0mM Zn2 TETA
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Conclusions

• Histidine rich proteins were successfully bound
  to Zn-phosphate and Zn-silica nanoparticles.
• Due to release of bound proteins after the
  addition of stronger chelating agent or imidazole
  or by lowering the pH, we assume that
  coordinative bond is present.
• Prepared Zn-phosphate nanoparticles were uniform
  in size around 50 nm.
• Released TNF-alpha analogues retained their
  biological activity reversible binding.
• The release from inorganic nanoparticles was
  achieved under very mild conditions during 2
  weeks close to phisiological conditions (pH
  around 6,0 or citrate present in 0,5 mM
  concentrations) formulations with prolonged
  release.
• Controlled protein self-assembly was achieved
  with the addition of Zn-ions and Zn-ions and two
  different chelating agents.

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Thank you for your attention!