L1 - Biomaterials: Foundations

1. Biomaterials Vocab
a. Synthetic or natural material suitable for use in concstructing artificial organs and
prostheses or to repair or replace damaged organ or tissue
i. Why?: treat disease, abnormalities, replacements
b. Ductility: ability to deform plastically before breaking
c. Biodegradation: degradation by biological molecules (enz or microbial deg)
d. Bioerosion: Erosion of a polymer into water soluble products under physiological
conditions
e. Bioabsorption/Bioresorption: products removed by cellular or physiological
activities
2. Types:
a. Metals
i. Strong, tough ductile/may corrode
ii. Metallic bonds, cloud of electrons/Fcc,hcp,bcc
iii. Degrade: oxides, biological fluids: atoms ionize and go into solution
iv. Pourbaix diagram; pH affects
v. Stress Corrosion cracking
1. Ex: Stainless steel,Cobalt/Chromium Alloys, Titanium Alloys
b. Ceramics
i. Strong in compression, stable and inert/brittle, weak in tension
ii. Types
1. Metal oxides
2. Calcium phosphates
3. Glasses and glass-ceramics
4. Carbon-based ceramics
c. Polymers
i. Easy to fabricate,versatile/not too strong and deforms
ii. Combosed of monomers and covalent bond along backbone, Van der
Waals forces between chains
iii. Addition polymerization: no by-products, monomers are doubles bonds or
rings, reactive end group
iv. Condensation polymerization: random union of monomer and oligomer
v. Stereochem: atactic, isotactic, syndiotactic
vi. Physical struct: linear, branched, crosslinekd, star, comb, dendrimers
vii. Types:
1. Crystalline: opaque (like Teflon); compact struct
2. Amorphous: glassy, randomly oriented, rubbery
viii. Thermal Transitions
1. Glass (Tg): undergoes transition from hard to rubbery
2. Melt (Tm): all cryst disappear (increase crys, increase Tm)
ix. Bulk Properties
1. Thermoplastics (linear and branched); soften/flow with temp and
pressure, reprocessing
 2. Thermosets (crosslinked): no reprocess, swellable
x. Example: PLGA: copolymer of PLA and PGA - bioresorpable; natural:
polysacc and GAGs (chitosan, agarose)
d. Composities
i. Combination of 2+ materials
ii. Strong, combine advantages/difficult to make
iii. Particulate, fiber, laminar

L2 - Biomaterial Interactions
1. Ceramics
a. Crystal Structures
i. Rock Salt structure: NaCl, MdO, MnS, LiF, FeO
ii. Fewer slip planes: ions make it less slip
2. Bioceramics
a. Types
i. Dense, nonporous, nearly inert (morphological fixation)
1. Al2O3
ii. Porous inert implants with bone ingrowth (biological fixation)
1. HA coated metals
iii. Dense, nonporous, surface creative ceramics (bioactive fixation)
1. Bioactive glasses
iv. Dense, nonporous/porous( resorbable) ceramics
1. Calcim suflate
b. More bioreactive = higher percentage of interfacial bone tissue earlier on
c. Sequence of interfacial reactions bn tissue and bioactive glass
i. Formation of SiOH bonds and Si(OH)4
ii. Polycondensation of SiOH+SiOH = Si-O-Si to form hydrated silica gel
iii. Adsorption of amorphous Ca+PO4+CO3
iv. Crystallization of hydroxyl carbonate apatite (HCA)
v. Adsorption of biological moieties in HCA layer
vi. Action of Macrophages
vii. Attachement of Osteoblast stem cells
viii. Differentiation and proliferation of osteoblasts
ix. Generation of matrix
x. Crystallization of matrix and growth of bone

L3 - Cell-ECM interactions
1. ECM
a. Native ECM
i. Structural and functional, facilitates signal transduction between adjacent
cells and between cells and ECM, fluctuates in response to environment
b. ECM based scaffolds
i. Decellularization of dermis, urinary bladder, SIS
2. ECM Components
 a. Collagen
i. Type 1 collagen structural. Other (type III) increased flexibility
b. Fibronectin
i. Promote adhesion to various cell types
ii. RGD (Arg-Gly-Asp) subunit is cell-ECM binding via integrin system
1. Integrins transmembrane on cell surface bind to RGD
c. Proteoglycan
i. GAG chains attached to core proteins. Net negative charge so it attracts
water
d. Laminin
e. Elastic Fibers
f. Fribril Associated CollagenFibrillar collagen
g. Integrin
h. Glycosaminoglycans (GAGs)
i. Polysaccharides that carry amine groups
ii. Bind growth factors and cytokines, promote water retention
3. Interactions:
a. Physical reordering:
i. Remodeling of ECM, degradation by Matrix Metalloproteinases (MMPs)
ii. Lysyl oxidase (LOX) affects crosslink of collagen and elastin
b. Stiffening
i. More focal adhesions are made. Stress fibers in specific direction
ii. Post-translational modification can regulate
iii. Proteoglycans and hyaluronic acid = hydration which affects compression
4. Cell Adhesion
a. Integrins
i. Transmembrane. Binds cell-cell (cadherins and selectins) and cell-
ECM(fibronectin, collagen, and laminin)
ii. Alpha and Beta subunits
iii. Effect of RGD density on cell adhesion and spreading (more dense =
more circular)

L4 - Biomaterial Surface Modifications

1. Why/How?
a. ECM components deposited on the surface, specific sequences engineered on
surface
b. Engineer biofunctionality to modulate biological response without affecting bulk
properties
2. Types:
a. Unmodified surface
b. Overcoat
i. Solvent coat, grafted surface layer, metalization, plasma deposit, sprayed
hydroxyapatite
ii. Plasma spraying: plasma jet droplets coate the substrate surface
 1. Clean, sterilize, coat, crosslink functional groups to surface
2. Atmospheric plasma treatment is ideal for in-line processing
iii. Solvent coating: dipped, sprayed, or rolled on substrate, simple coating,
can control drug release by retarding drug diffusion
c. Surface gradient
i. Graft, interpenetrating network, ion implant
d. Self-assembled film
i. Thiols on gold, silanes on silica, phosphates on Ti, multilayers are
possible
ii. Sponaneously formed, amphiphilic (polar and nonpolar parts)
1. Functional head, assembling alkyl chain, anchor group
2. Van de Waal’s forces between chains = stable structure
iii. Exothermic reaction
e. Surface active bulk additive
i. Low molecular weight, polymeric
f. Surface chemical reaction
i. Oxidation, fluorination, silanization
g. Etching and roughening
i. Surface chemical compositional changes are frequently observed
h. Polyelectrolyte multilayer films
i. Polyelectrolytes, proteins, nanoparticles
ii. Polyelectrolyte: alternate adsorption of polycation and polyanion
monolayers on polymer surface - can incorporate components into the
layers
3. Modified Titanium implants
a. Plasma treatment had higher bone pull-out force
4. Hyaluonic acid/chitosan polyelectrolyte multilayer
a. Promote osteoblast functionsand inhibiting bacterial adhesion
b. Chitosan (CH) has bactericidal nature via alter bacterial cell membrane’s
permeability via interaction of pos charged amin groups with negatively charged
cell membrane
5. SAMs with different functional groups coated with fibronectin
a. NH2 had highest differentiation
6. PTFE (poly[tetrafluoroethylene])
a. Attach to virtually all inorganic and organic surfaces
b. DOPA - plaque-substrate interface with strong covalent and noncovalent
interactions
7. PEG:
a. Prevents protein adsorption and cell attachment, volume of surface taken over by
bulky chains
8. Parylene coating
a. Electrical insulation, posture and gas barrier properties
b. Implanted electrodes and implanted circuitry
9. Heparin as anticoagulant
 a. SS surface modified with plasma then Heparin immobilized

L5 &6 - Biomaterial Surface patterning and cell behavior and Topography

1. Topography and contact guidance - Introduction
a. “The orientation of cell and stress fibers is influenced by the geometrical patterns
such as nano/microgrooves on substrates, or collagen fibers in gens and soft
tissues”
b. Native tissues
i. Blood vessel: smooth muscle, collagen, endothelial coating
ii. Corneal epithelial basement membrane: all clustered
iii. Heart: all aligned myocardium
c. Cell behavior and responses:
i. Features: size, shape, geometrical arrangement, distance between
topographical features
ii. Responses: adhesion and alignment, morphology, migration,
differentiation, proliferation
2. Types and limits of topographical cues
i. Micro-topography (10um) = whole cell morphology
ii. Nano-topography = subcellular sensing mechanisms
a. Focal adhesion complex:
i. Integrin containing, connects cytoskeleton to ECM
b. RGD spacing and cell adhesion
i. Adhere <70nm better
c. Other topo features
i. Fibroblast respond to 27nm high nano-islands
3. Topographic features and their effect on cell behaviors
a. Elongation and Alignment
i. hMSC elongation along 250nm width nanogratings (~100nm critical
depth)
ii. Fibroblasts aligned themselves on grooves depths as shallow as 35nm
(~35 nm: critical depth)
iii. Transition from micro to nanoscale = trans from paralel to perpendicular
(pitch)
4. Applications of contact guidance in tissue engineering
a. Neurite guidance:
1. Growth cone used to sense and navigate the surrounding
environment: 10um2-760um2
2. Deeper gratings makes the neurite have to bend to reach the
bottom = doesn’t extend as much in that direction
3. NPCs - TUJ1 positive
ii. Inducing differentiation - Dopaminergic neurons
1. Nanogratings able to bias differentiation without any other
biochemical factors
iii. Competitive stimuli
 1. Neuronal polarization modulated by biochemical cues (laminin),
NGF, and topography
2. Physical stimuli beat chemical - strong effect on polarization
b. Engineering Muscle Fibers
i. Mimicking: highly oriented myofibers formed from mononucleated muscle
cells
ii. Contact guidance: electrospinning PCL/collagen as a scaffold
c. 3D topography
i. PA hydrogel around lattice of glass beads. Glass beads dissolved leaving
3D network = can make spheroids
1. Is nonfouling = cells adhere to each other instead of the scaffold =
spheroid

L7 - Surface Characterization Technology

1. Roughness/Topography
a. Atomic Force Microscopy (AFM)
i. VdW/Electrostatic forces between tip and sample deflelcts to give a
difference in height
ii. Tip can be functionalized to be more attracted to a specific tag
1. Made from silicon
2. Top labeled with Ni2+-NTA to HIS-tagged proteins or use a ligand
iii. Tapping Mode, Contact Mode, Non-contact mode
1. Phase shift could be due to adhesive, stiffness, or friction
b. Scanning electron microscopy (SEM)
i. 3-100nm resolution. Focus electron lasers for emission of electrons.
Measure intensity of secondary electron emission on a detector
2. Chemical Composition
a. Fourier transform infrared spectroscopy (FTIR)
i. Bonds absorb wavelengths and vibrate. Infrared spectrum has peaks
corresponding to frequencies of vibrations - can determine which peaks
respond to which bonds

ii.
b. X-ray photoelectron spectroscopy (XPS)
c. Auger electron spectroscopy (AES)
d. Secondary ion mass spectroscopy (SIMS)
e. Energy dispersive X-ray spectrometry (EDS)
3. Surface energy/wettability
a. Contact angle goniometry
i. Angle indicates degree of wetting: 3 interfaces (L-V, S-V, S-L)
 ii. Energetics at each interface results in a certain degree of spreading
4. In-Vivo
a. Micro-computed tomography (micro-CT)
i. Takes 2D xrays of the material and layer them to make a 3D image

L8 - Microparticles and Nanoparticles

1. Endocytosis of particles into the cell
a. Vocab:
i. Pinocytosis: ingestion of liquid
ii. Receptor mediated endocytosis: special receptor proteins
iii. Phagocytosis: membrane diffusion/disruption
2. PEG-Based Nanoparticles
a. Adv: ease of fabrication, encapsulate many things, intracellular delivery
b. Dis: degradation can by bio incompatible, potential immune response
3. Materials: Lipids, polymers, surfactants
a. Microspheres made from chitosan and alginate are common
i. Alginate beads-cell encapsulation of pancreatic islet cells
1. Facilitates diffusion of insulin while excluding immune cells that
could reject the encapsulated cells
2. Z1-Y15 alginate: triazole-contianing alginate analogues (FBR
evading material) - reduces macrophage activation
4. Uses:
a. Therapeutics (small molecules, proteins) delivery
b. Delivery and maintenance of transplanted cells
c. Delivery of DNA and RNA
i. Has to overcome: degradation, poor uptake, toxicity
ii. siRNA mechanism of gene silencing (siRNA duplex => formation of RISC
-> siRNA/mRNA-complex = silenced mRNA)
iii. Disrupt the endosome or escape disruption
iv. Multicomponent microparticles: loading both siRNA and pDNA in a single
particle
v. IL-10 targeted siRNA => IL-10 targetted to T cells
5. Microparticle preparation techniques
a. Single and double emulsion solvent evaporation
i. Drug encapsulation

ii.
b. Spray drying
i. Polymer solution with drug sprayed through nozzle
ii. More porous particle (dry powder)
 c. Ionic gelation
i. Used with chitosan and alginate (chitosan disrupts the charge of the
membrane of the cell), put drops into polymer solution with continuous
stirring

L9 - Microparticles Continued (Applications)

1. Why are they relevant?
i. Therapeutics delivery, maintaining transplanted cells, delivery of DNA and
RNA, Multifunctional nanocarriers, scaffolds
a. Scaffolds
i. Multiphased scaffold = better interface for soft-tissue to bone integration
(ACL to bone integration)
1. Phase A: Acellular and PGA (ligament – fibroblasts and soft
tissue)
2. Phase B: PLGA microspheres, sintered
(fibro-condrocytes/cartilage)
3. PLGA and bioglass composite microspheres, sintered
(osteoblasts)
ii. “Stratified Scaffold” distincted yet continuous mult-tissue regions.
iii. PLGA microspheres are rigid in shape and can be packed together alone
or with other materials = porous 3D structures
2. What factors determine their interaction with cells?
a. Particle Shape affects interaction (not been fully explored)
b. Nonspherical particles generated via: microfluidics (rods and discs), self-
assembly, direct replication, photolithography, soft lithography
c. Requirements for Producing Drug Carriers:
i. Must be applicable to polymers
ii. Must allow for incorporation of theraputic drug molecules while not
inactivating drug
iii. Shape method should not inhibit post-production processing
3. Complex Shape example: PS and PVA
a. Started with spherical polystyrene (PS) particles and suspended in polyvinyl
alcohol (PVA) being cast and manipulated into a new shape
i. Sheme A: by liquefying using solvent or heating of PS and then stretch
them
ii. Sheme B: PVA films are stretched creating voids around the particle.
Then voids are filled by liquifying the PS
b. Particle shape dictated by: material properties of film (Tg and thickness),
particles (Tg and viscosity), interaction between particles and film (adhesion),
and stretching parameters
i. Volume remained constant = size and shape controlled independently
4. Particle shape in Drug Delivery:
a. Degradation profiles could be unique and the shape of the particle will change
over time
 b. Transport will be different
c. Influences targeting ability: overall SA and local curvature effects ligand binding
d. Ability for cells to internalize particles
5. Target geometry in phagocytosis
a. Local shape determines complexity of actin structure needed to initiate
phagocytosis. If the required actin fails then only simple spreading occurs
b. Actin cup forms beneath the particle which becomes an actin ring
i. Spheres and EDs attached at major axis had shorter actin cup times. For
flat side of EDs: only contact and spreading, no cup
6. Mammalian cells preferentially internalize hydrogel nanodiscs over nanorods
a. Parameters: contact/adhesion force between nanoparticle surface and cell
membrane (shape); sedimentation (weight), strain energy required for membrane
deformation (shape)
7. Cellular hitchhiking by nanoparticles
a. Avoid immune clearance and up circulation time => use circulatory cells like
RBCs and immune cells - 110-1100nm and surface functionalities can adsorb to
RBCs (carboxyl, anime, aldehyde)
b. Non-covalent adsorption on erythrocytes
c. Targetting using Circulatory:
i. RBCs: adsorption via hydrophobic, electrostatic, hydrogen bonds, and
VdW reversibly attaches PS (non-covalent bonding)
ii. Receptor-Ligand: HA (ligand) attached to CD44 (receptor) of
macrophages, T cells and B cells
iii. Covalent conjugation: thiol rich T surface directly conjugated with
maleimide liposomes

L10 - Nanoparticles in Bioimaging

1. Gold (Au) nanoparticles: AuNPs
a. Reasons they are popular:
i. High chemical and physical stability. Biocompatibility of gold
nanostructures
ii. Ease of surface functionalization with organic and biological molecules
iii. Optical properties: surface plasmon resonance
1. Different colors depending on the size and shape. Resonance
when electrons on material vibrate at same frequency of light =
color
b. Synthesis

i.
 ii.Citrate (or sodium borohydrate or ascorbic acid) for reduction and
stabilization
iii. Thiol groups for surface functionaliation (SH)
c. Anti-sense oligonucleotide capped gold nanoparticles Covid 19 detection assay
i. Agglomeration of AuNPs a redshift of ~40nm from violet to dark blue
ii. RNase H can recognize and cleave the Covid-19 RNA strand when it is
hybridized with Au-ASO
iii. More agglomeration and the AuNPs precipitate out with attached RNA
strand which we can see
2. Quantum dots (QDs or QDots)
a. Characterize
i. Semiconducting material with dia = 2-10nm
ii. Unique electronic properties, intermediate between bulk semiconductors
and discrete molecules/atoms
iii. Fluorescence using distinctive colors determined by particle size
b. Advantages
i. QD light absorption = electron being excited and leave behind a hole.
This hole and electron bind = exciton then the electron comes back to
hole and light emitted
ii. Size of QD decreases = E difference between highest and lowest
conduction bands increase = color shift from red to blue (larger to smaller
QDs)
iii. Optical properties modulated easily use of different colors
iv. More photostable (Better than Alexa 488) and better signal to noise ratio
(resistant to photobleaching); Co-staining of cellular components
c. Types
i. Core-Type QDs:single component materials like chalcogenides (slenides,
sulfides, tellurides) of cadmium, lead, or zinc: Optical by changing size
ii. Core-Shell QDs: one material embedded (CdSe core and ZnS shell).
Coating material semiconductor with wider band gap which improves
quantum yield = more efficient and bright
iii. Alloyed QDs:alloying two semiconductors with different badn gap
energies (finetune properties without changing size)
d. Examples: easily taken up by the body = In vivo cancer targeting and imaging
i. Core=shell CdSe-ZnS QDs protected by coordinating ligant (TOPO)
attached to amphiphilic polymer coating while having sites for PEG and
ligand attachment; triblock polymer coating for preventing aggregation.
Ligands for antigen recognition and PEG for biocomp
ii. Optical properties did not change in a pH 1-14 or salt (0.01-1M)
iii. Via EPR (passive) and antibody conjugates (active)
1. PSMA Ab (prostate specific membrane antigen) for labeling
specificity
iv. No localized fluorescence signals in healthy mouse even with same
amount of QD injection
 v. Retention and distribution in 6 normal host organs
1. With excess COOH groups: no tumor targeting = rapid clearance
by reticuloendothelial system (RES)
2. RES is hetoerogeneous population of phagocutic cells in various
tissues - clear particles and soluble substances
e. Limitation
i. Since they are metals need to think about the biocompatibility!
ii. Large photoluminescence (PL) line width and narrow bands are
associated with more toxic (Cd) QDs
1. Showed narrowing PL emission with epitaxal coating of CulnS2
core with a “thicker” ZnS shell

L11 - Stimuli-responsive Material

- Biomedical strategies lean towards “multi-pronged” combination approaches for
diagnostics
1. Characteristics
a. Respond to slight phs/chem change with sharp property changes “Smart
Or “Environmentally sensitive”.
b. Incorporate stimulus-responsive groups = material transformations
c. Property Changes: (physical, chemical, biological)
i. Reversible precipitation or gelation
ii. Reversible adsorption on a surface
iii. Reversible collapse of surface graft polymer
iv. Reversible collapse of hydrogel
2. Physically Responsive
a. PNIPAAm LCST (exibits lower critical solution temp) of 32C
i. Loss of bound water to bulk solution and becomes insoluble in water
(hydrophobic)
ii. Cultured fibroblasts on it and were able to detach cell sheets (monolayer)
from the substratum by just lowering the temperature without trypsin or
EDTA
1. Detached sheets formed multicellular spheroid
iii. Cell sheets:
1. Can be transplanted without biodegradable scaffolds or sutures
2. Thick tissue constructs and patterned sheets with 2+ sources are
also able to make layered sheets.
iv. Wound dressing:
1. 3 sections: non-waoven polypropylene material/PNIPAAm
hydrogem/Tri-copolymer sponge
v. Patterned cell sheets and LCST modulation
1. NIPAAm co-poly with hydrophilic polymers (Acrylamide): increase
LCST
2. NIPAAm polymerized with hydrophobic polymers: decrease LCST
 3. Mimic = need spacially ordered tissue architectures with
heterotypic cell-cell interactions… not easy to do because
adhesion and proliferation not the same
4. Patterned Sheet: PNIPAAm-BMA in PNIPAAm

5.
vi. Monomer NIPAAm exhibits some cytotoxicity
3. Chemically Responsive: Light Responsive
a. Free radical polymerization when exposed to UV light
b. “Transdermal potopolymerization for minimally invasive implantation”
i. Polymer hydrogel system (PEO) - PEO dimethacrylates
ii. Testing concept of transdermal polymerization
iii. Applyed to tissue via injectable cartilage model system. Implants
harvested demonstrated collagen and proteoglycan production/histology
comparable to native neocartilage
c. Challenges:
i. UV exposure may be damaging to cellular DNA insurrounding tissue
ii. Synthetic photopolymerizable hydrogels do not contain cell binding motifs
and are not biodegradable
1. Could use natural/hybrid hydrogels but limited work so far
4. pH sensitive polymers
a. Helpful for drug targetting (pH varies in body and polymer bonds are pH labile
b. Lysosomes have acidic pH
c. pH responsive Polymers for the Intracellular Delivery of Biomolecular Drugs
i. “Encrypted Polymers” 3 primary Functionalities:
1. A targeting agen that directs receptor-mediated endocytosis
2. pH-responsive element selectively disrupts endosomal membrane
3. Biomolecular therapeutic component which is delivered as a free
and active agent into the cytoplasm
d. Mimic design of viruses target and direct cellular uptake and enhance cytosolic
delivery (disrupt endosomes)
i. Remain PEGylated at pH 7.4, rapid de-PEGylated after endocytosis when
endosome pH~5
ii. DMAEMA and hydrophobic BMA with BA make up membrane-disruptive
backbone (alkyl groups)
iii. Acid-degradable acetal bonds = pH sensitivity that link the PEGs and
PEGylated drugs/targetting ligand

L12 - Stimuli-Responsive Material

 - Enz cross-linked hydrogels via horseradish peroxidase (HRP)-catalyzed cross
linking
- Crosslinking reaction occurs upon mixing phenol-rich polymers with HRP and
hydrogen peroxide in aqueous solution
1. Enzyme-catalyzed in situ forming gelatin hydrogels as bioactive wound dressings:
effects of fibroblast delivery on would healing efficacy
a. Gelatin-hydroxyphenylpropionic acid (GH) hydrogel via HRP-catalyzed
crosslinking: good biocompat
i. Hydrogel showed strong tissue adhesion due to enz xlinking between
phenol gelatin and tyrosine tissue
ii. Softer hydrogel = more spreading morphology and faster deg rate
b. Assess wound healing of DFBs plus GH hydrogels showed helped with formation
of new microvessels and reconstructed dermis tissue
2. Dual Stimuli
a. Insulin delivery triggered by glucose
i. Continuous glucose-monitoring sensor with external insulin infusion pump
b. Glucose responsive gel
i. Glucose oxidase (GOx/GOD) catalyzes glucose to gluconic acid
ii. Trapped in pH sensitive matrix. Presence of glucose = decrease pH =
matrix volume increases and releases insulin
iii. Challenges: slow response rates to changes in glucose concentration,
low membrane mechanical strengh = leaking and risk of hypoglycemia,
enzyme denaturation, immunogenicity, low loading capacity
3. Glucose Responsive Microgels Integrated with Enzyme Nanocapsules for closed loop
insulin delivery
a. Microgels encapsulate insulin and enzy nanocapsules. Three Components
i. pH responsive polymeric materix
ii. Gox- and catalase (CAT)- containing enzyme nanocapsules
1. CAT added to consume H2O2 which may be toxic and deactivate
GOx
iii. Human recombinant insulin
b. Microgels were made of Chitosan by elecrospraying to get monodispersed
particles
i. Swelling of microgels and release of insulin dure to rich amine groups
increasing the charge in gel matrix
1. Reversible under normoglycemic conditions (migrogel shrinks and
release decreases)
ii. Tripolyphosphate (TPP) used to cross-link chitosan matrix and entrap
nanocapsules and insulin
1. Nanocapsules mixed with chitosan and sprayed at TPP at high
voltage for cross-linking
iii. Process:
1. Nanogels were encapsulated with GOx or CAT
2. Vinyl groups covalently conjugated to enz
 3. Nanocapsules prevared via free radical polymerization with
acrylamide, APMAAm and cross-linker
iv. Nanocapsules:

1.
v. Responsiveness:
1. Insulin release measured at different glucose concentration
2. Invivo: maintained in normoglycemic range for 12h and gradually
increased afterward (22 days)