Nanobiotecnologia MG1

8/3/2019 Nanobiotecnologia MG1

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NanobiotecnologiaNanomedicinaBiomolculasBionanoestruturas

Miguel GamaInstitute for Biotechnology and BioengineeringMinho UniversityPortugal

There is plenty of room at the bottom Richard Feyman, 1959


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Continued growth in Healthcare, largest service sector

NanoMedicine is about early diagnosis, early andregenerative treatment

Through screening and early diagnosis find diseaseearly and intervene early

European Technology Platform on Nanomedicine

Industry and academia together with the European Commission identified the need for a European initiative to intermesh the severalstrands of nanomedicine in order to create a firm strategy for

advancement.

The resulting European Technology Platform on NanoMedicine isan industry-led consortium

October 2006: Strategic Research Agenda (SRA) - breakthroughdevelopments in the areas of diagnosis, targeted delivery systems,and regenerative medicine .


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DiseasesCardiovascular Diseases

Cancer

Musculoskeletal DisordersNeurodegenerative Diseases and

PsychiatricConditions

Diabetes

Bacterial and Viral Infections andDiseases

1. Nanotechnology based Diagnostics and Imaging2. Targeted Drug Delivery3. Regenerative Medicine

NanoDiagnostics: early and accurate diagnosis- Biosensors and miniaturized devices- targeted imaging agents to highlight of disease

Nano DiagnosticsOpportunities Screening: personal risk factorsIdentification of population at riskPrediction of risk factors

Earlier, more sensitive, faster diagnosticDiagnosis of asymptomaticpatientsHigher sensitivity: detection of early biomarkersNon- invasive and painless diagnostic techniques


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In-vivo Nano-imagingTumor blood vessels have several abnormalities compared with

physiological vessels like relatively high proportion of proliferatingendothelium cells, an increased tortuosity and an aberrant basementmembrane formation. The rapidly increasing tumor vasculature often

has discontinuous endothelium, with gaps between the cells thatmay be several hundred nanometres large.

1. Nanotechnology based Diagnostics and Imaging

2. Targeted Drug Delivery3. Regenerative Medicine

Synthetic nanometer- size drug delivery systems for delivery of therapeutic agents and biologically active products being ableto tackle challenging diseases.

The development of targeted delivery is firmly built onextensive experience in pharmaco-chemistry, pharmacology,toxicology, and nowadays is being pursued as a multi- andinterdisciplinary effort.


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NanocarriersNanoparticleshave in general relatively higher intracellularuptake.Nanoparticlescan penetrate the submucosal layers while themicroparticlesare predominantly localized in the epithelial lining.Nanoparticlescan be administered into systemic circulationwithout the problems of particle aggregation or blockage of fineblood capillaries.

1. Nanotechnology based Diagnostics and Imaging2. Targeted Drug Delivery

3. Regenerative Medicine

Regenerative medicine has thecapability to radically change theway some diseases are managedin the future.By leveraging novel cell culturetechniques and the design of bioresorbable polymers, tissueengineering strategies haverecently emerged as the most

advancedtherapeuticoptionpresentlyavailableinregenerativemedicine.

ChallengesInitiate and control regeneration of pathological tissueTreat, modify and preventdisablingchronic disorders suchas osteoarthritis and diseases of the cardiovascular and centralnervous system.


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Regenerative MedicineIn the field of biomaterials and biotechnology, the term'biomimesis' has been established to describe the process of simulating what occurs in nature.

The biomimeticphilosophy can be condensed into three basicelements:IntelligentbiomaterialsBioactive signaling moleculesCells


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BiomoleculesAminoacids and proteins


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Glycine (Gly, G)

Threonine (Thr, T)

Leucine (Leu, L)

Proline (Pro, P)

Alanine (Ala, A)

Isoleucine (Ile, I)

Serine (Ser, S)

Methionine (Met, M)

Valine (Val, V)

Cysteine (Cys, C)


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Aspartic acid (Asp, A)

Tryptophan (Trp, W)

Phenylalanine (Phe, P)

Arginine (Arg, R)

Glutamine (Gln, Q)

Glutamic acid (Glu, E)

Histidine (His, H)

Tyrosine (Tyr, Y)

Asparagine (Asn, N)

Lysine (Lys, K)

R-COOH R-COO - + H+R-NH 3+ R-NH 2 + H +

Alanine titration


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Propriedadesdos aminocidosPotencialpara ligaes de hidrognio, reactividade

Exclusivamente dadores de H

Aceitadores e dadores de H

Aceitadores ou/e dadores de H(dependendo do pH)

Individual properties of the aminocids

amino acid masssurface b volume c pKa d pI

e solubility e density e

Alanine ALA A 71.09 115 88.6 - 6.107 16.65 1.401

Arginine ARG R 156.19 225 173.4 ~12 10.76 15 1.1

Aspartic Acid ASP D 114.11 150 111.1 4.5 2.98 0.778 1.66

Asparagine ASN N 115.09 160 114.1 - - 3.53 1.54

Cysteine CYS C 103.15 135 108.5 9.1-9.5 5.02 very high -

Glutamic AcidGLU

E129.12

190 138.44.6

3.08 0.864 1.460

Glutamine GLN Q 128.14 180 143.8 - - 2.5 -

Glycine GLY G 57.05 75 60.1 - 6.064 24.99 1.607

Histidine HIS H 137.14 195 153.2 6.2 7.64 4.19 -

Isoleucine ILE I 113.16 175 166.7 - 6.038 4.117 -

Leucine LEU L 113.16 170 166.7 - 6.036 2.426 1.191

Lysine LYS K 128.17 200 168.6 10.4 9.47 very high -

Methionine MET M 131.19 185 162.9 - 5.74 3.381 1.340

Phenylalanine PHE F 147.18 210 189.9 - 5.91 2.965 -

Proline PRO P 97.12 145 112.7 - 6.3 162.3 -

Serine SER S 87.08 115 89.0 - 5.68 5.023 1.537

Threonine THR T 101.11 140 116.1 - - very high -

Tryptophan TRP W 186.12 255 227.8 - 5.88 1.136 -

Tyrosine TYR Y 163.18 230 193.6 9.7 5.63 0.0453 1.456

Valine VAL V 99.14 155 140.0 - 6.002 8.85 1.230


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Hydrophobicity scales a Residue non-polarsurface area b

[A2]

Estimated hydrophobiceffect

for residue burial[kcal/mol]

Estimated hydrophobiceffect

for side chain burial c[kcal/mol]

Gly 47 1.18 0.0

Ala 86 2.15 1.0

Val 135 3.38 2.2

Ile 155 3.88 2.7

Leu 164 4.10 2.9

Pro 124 3.10 1.9

Cys 48 1.20 0.0

Met 137 3.43 2.3

Phe 39+155 3.46 2.3

Trp 37+199 4.11 2.9

Tyr 38+116 2.81 1.6

His 43+86 2.45 1.3

Thr 90 2.25 1.1

Ser 56 1.40 0.2

Gln 66 1.65 0.5

Asn 42 1.05 -0.1

Glu 69 1.73 0.5

Asp 45 1.13 -0.1

Lys 122 3.05 1.9

Arg 89 2.23 1.1

R

H2

C

N2

O 2

C2

N1

C1

O1

H

H1

C

C - t erminal

N - terminal

Ligao peptdica

ProtenasEstrutura primria

Estrutura secundriaEstrutura terciria

Estrutura quaternria


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Estrutura secundriaEstrutura terciria

Estrutura quaternria

-helix -sheet


Estrutura secundria

Estrutura terciriaEstrutura quaternria

H 188S 120

D 175

Hemoglobine

IL-10

Cutinase

- Enzimas (catalisadores)- Receptores (protena G - transduo de sinal)- Hormonas (insulina, )- Transportadores (albumina humana)- Estrutura (matriz extracelular, .)......


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BiologicnanoporesThe nicotinicreceptors of actylcholine is a ionotropic receptor that control the

synapsis at the nervous system, transiently increasing the permeability to ions atthe post-synaptic membrane. Acetylcholine(at the neuromuscular junction) and

glutamate (at the CNS) increase the permeability to the sodiumand potassiumions

Engineered nanoporesAlpha-hemolysin, a self-

assembling bacterial toxin

The -barrel pore-forming toxins(PFTs) are a family of polypeptides that are related

by sequence and structure.PFTsare secreted by bacteriaas water-soluble monomers,which bind to the surfaces of susceptible cells and assembleinto transmembrane poresleading to cell permeation andlysis (inner diameter of pore~2nm).


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Stochastic Sensors - are created by placing a nanometre-sized pore in aninsulating membrane and measuring the ionic transport through the pore in the

presence of the molecules of interest. The magnitude, duration, and rates of occurrence of the resultant current blockades allow rapid determination of analyte

concentration as well as discrimination between similar molecular species .

http://www.ks.uiuc.edu/Research/hemolysin/

Suspended in a lipid bilayer, an alpha-hemolysin channel becomes a stochasticsensor when a molecular adapter is placed inside its genetically re-engineered stem,

influencing the transmembrane ionic current induced by an applied voltage bias.Reversible binding of analytes to the molecular adapter transiently reduces the ioniccurrent. The magnitude of the current reduction indicates the type of analyte, whilethe frequency of the current reduction intervals reflects analyte concentration. Such

stochastic sensors were demonstrated to simultaneously measure, with a singlesensor element, concentrations of several organic analytes.


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The stochastic sensing of smallorganic molecules was a substantialchallenge that was solved by theintroduction of noncovalentmolecular adapters. Adaptermolecules, such as cyclodextrins,can become lodged in the lumen of the HL pore, where they remainavailable for the hostguestinteractions. Hence, binding of acyclodextrin within the lumen of the HL pore causes a reduction incurrent flowing through the pore.Mutant homoheptameric HL pores(M113X) are capable of accommodating CD for tens of seconds. binding of a cyclodextrinwithin the lumen of the HL porecauses a reduction in currentflowing through the pore.Additional transient reductions inthe current are brought about bythe subsequent binding of organicanalyte molecules.


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DNA sequencing - The transmembrane pore of alpha-hemolysin can conduct notonly small solutes, but also rather big (tens of kDa) linear macromolecules. Thus,

driven by a transmembrane potential, DNA or RNA strands can translocate throughthe pore of alpha-hemolysin, producing the ionic current blockades that reflect thechemical structure of individual strands (). Statistical analysis of many such blockage

currents allowed the researchers to discriminate different sequences of RNA andDNA homopolymers, as well as the segments of purine and pyrimidine nucleotideswithin a single RNA molecule. A single nucleotide resolution has been

demonstrated for DNA hairpins, raising the prospect of creating a nanopore sensorcapable of reading the nucleotide sequence directly from a DNA or RNA strand.

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Nanobiotecnologia MG1