nanotechnology I, II, III - minko

types of cancer

each organ in the blood is composed of different types of tissue, and most cancers arise in 1 or 3 main types → epithelial, connective or blood forming tissue

• carcinomas = cancer that occur in epithelial tissues

  • skin, inner membrane surfaces of the body (such as those of the lungs, prostate, breast, ovarian, stomach, intestines, blood vessels)

• sarcomas originate in connective tissue

  • muscle, bone, cartilage, fat

  • parts that support and connect other parts of the body

• leukemias develop in blood cells and lymphomas originate in the lymphatic system

• ranking of prevalence

  • carcinomas ~90%

  • leukemias + lymphomas ~8%

  • sarcomas ~2%

nanotechnology

• nanotechnology is a field of applied science focused on the design, synthesis, characterization, and application of materials and devices on the nanoscale

  • focused on creation and the use of materials and devices at the level of molecules and atoms

• nanomedicine = offshoot of nanotechnology, refers to highly specific medical intervention at the molecular scale for curing disease or repairing damaged tissues such as bone, muscle, nerve or cells

nanotechnology scale

• what is considered nanotechnology/nanomedicine

  • small dye molecule → 0.5 nm-10nm

  • fluorescent protein → 10 nm-20nm

  • virus → 20nm - 400 nm

• atoms are SMALLER than nanotech

• bacteria/animal cell are LARGER than nanotech

classes of nanoparticles

• polymeric

  • examples

    • polymersome

    • dendrimer

    • polymer micelle

    • nanosphere

  • advantages

    • precise control of particle characteristics

    • payload flexibility

    • easy surface modifcation

  • disadvantages

    • possibility of aggregation and toxicity

• inorganic

  • examples

    • silica nanoparticle

    • quantum dot

    • iron oxide nanoparticle

    • gold nanoparticle

  • advantages

    • unique electrical, magnetic, optical properties

    • variability in size, structure, geometry

    • suited for theranostic applications

  • disadvantages

    • toxicity and solubility limitations

• lipid-based

  • examples

    • liposome

    • lipid nanoparticle

    • emulsion

  • advantages

    • formulation simplicity

    • high bioavailability

    • payload flexibility

  • disadvantages

    • limited encapsulation efficiency

dispersed systems

dispersed systems consist of particulate matter known as dispersed phase distributed throughout a continuous or dispersion medium

• dispersed material may range in size from particles of atomic and molecule dimensions to particles whose size is measured in millimeters

• dispersed systems are classified based on the mean particle diameter of the dispersed material

3 size classifications

• molecular dispersion

  • size

    • <1 nm

  • characteristics of particles

    • invisible in electron microscope

    • pass through ultrafilter and semipermeable membrane

    • undergo rapid diffusion

  • examples

    • oxygen molecules

    • ordinary ions

    • glucose

• colloidal dispersion (IMPORTANT ONE)

  • size

    • 1 nm - 0.5 micrometers

  • characteristics of particles

    • NOT resolved by ordinary microscope (although may be detected under ultra microscope)

    • visible in electron microscope

    • pass through filter paper

    • do NOT pass semipermeable membrane

    • diffuse very slowly

  • examples

    • colloidal silver sols

    • natural and synthetic polymers

    • cheese

    • butter

    • jelly

    • paint

    • milk

    • shaving cream

    • nanoparticles

• coarse dispersion

  • size

    • > 0.5 micrometers

  • characteristics of particles

    • visible under microscope

    • do NOT pass thru normal filter paper

    • do NOT dialyze thru semipermeable membrane

    • do NOT diffuse

  • examples

    • grains of sand

    • most pharmaceutical emulsions and suspensions

    • RBCs

liposomes and drug delivery

liposomes can carry both hydrophobic molecules and hydrophilic molecules

• a liposome encapsulates a region on aqueous solution (core) inside a hydrophobic membrane. dissolved into the core, hydrophillic drugs can NOT readily pass thru the lipids

• hydrophobic drugs can be dissolved into membrane

intracellular drug delivery by liposomes: mechanisms of drug release

• diffusion

  • drug inside the carrier diffuses out where there is a change in pH

• membrane fusion

  • carrier fuses directly w/the plasma membrane, releasing the drug contents into the inside of the cell

• endocytosis

  • entire drug loaded carrier is engulfed by the cell thru endocytosis

antisense oligonucleotides (ASO) therapy

• typical pathway

  • DNA → transcription → mRNA → translation → protein → function

• ASO therapy is focused on the blockade of the synthesis of specific protein by binding to mRNA

antisense oligonucleotides (ASO)

• ASO = single strands of DNA or RNA that are complementary to a chosen sequence

mechanisms of antisense oligonucleotides action

• binding of ASO to mRNA forming a duplex and preventing the synthesis of specific protein

• enzymatic degradation of mRNA portion of the duplex which releases the ASO for further binding to another mRNA

• the release of ASO by the cell into the blood stream and excretion (usually unchanged) by the kidney

RNA interference (RNAi)

according to recent research, double-stranded RNA molecules (dsRNA) efficiently and specifically inhibits gene products of a post-transcriptional level (gene silencing) → this phenomenon, also called RNAi, has been found in numerous organisms and can be induced artificially

co-suppression

• formation of double stranded RNA (dsRNA) can inhibit gene expression in many different organisms

• first discovery of this inhibition in plants was more than a decade ago and occurred in petunias; researchers were trying to deepen the purple color of flowers by injecting the gene responsible for the color into petunias but were surprised at the result → instead of darker flower, petunias were either variegated or completely white

  • phenomenon = co-suppression since both the expression of the existing gene (initial purple color) and the introduced gene (to deepen the purple) were suppressed

• co-suppression has since been found in many to inhibit gene expression in other plant species and in fungi; double stranded RNA responsible for this effect

RNA interference and small interfering RNA (siRNA)

• since the only RNA found in a cell should be single stranded, presence of double stranded RNA signals = abnormality

• cell has specific enzyme (in drosophilia it is called dicer) that recongnizes the double stranded RNA and chops it up into small fragments between 21-25 BPs in length → these short RNA fragments (siRNA) bind to RNA-inducing silencing complex (RISC) → RISC is activated when the siRNA unwinds and the activated complex binds to the corresponding mRNA using antisense RNA → RISC contains an enzyme to cleave the bound mRNA (called slicer in drosophilia) → cause gene suppression → once mRNA has been cleaved it can NO longer be translated into functional protein

advantages of antisense/siRNA therapy

• extremely high specificity

• selective knockout of single critical target

• potential for increased efficacy

• reduced likelihood of side effects

small interfering RNA (siRNA) therapy: challenges

• low stability → siRNA backbone is susceptible to hydrolysis by serum nucleases in extracellular environment

• integrity of chemically stabilized siRNA is maintained in blood for at least 30 minutes whereas unmodified (naked) siRNA duplex is degraded within 1 minute

• rapid elimination → the small size of siRNA leads to the rapid elimination of naked, unmodified siRNAs from the circulation by renal clearance (circulatory half-life of siRNA < 5 mins)

• poor cellular internalization → delivery of siRNA across the cell membrane into the cells is limited by the repulsion of the negatively charged siRNA by the like-charged cell membrane

branched polymer: dendrimers

• dendrimer = regularly branched molecule which resembles the branches of a tree

• properties of dendrimers are dominated by the functional groups on the molecular surface:

  • can be water soluble when its end group is a hydrophilic group like carboxyl group

  • theoretically possible to design a water-soluble dendrimer with internal hydrophobicity which would allow it to carry a hydrophobic drug in its interior

  • volume of a dendrimer increases when it has a positive charge

polyamidoamine (PAMAM) dendrimers

greater the generation → greater molecular weight → greater diameter → greater surface groups

surface modified and internally cationic PAMAM dendrimers for efficient siRNA intracellular delivery

traditional dendrimers

• non-modified surface; external positive chargers for siRNA binding

• nano-fiber shaped structures (will ask about this)

modified dendrimers

• acetylated surface; internal positive charges for siRNA binding

• chemical modification of surface moves the + charge inside dendrimer

• well-condensed spheroidal nanoparticles (will ask about this)

surface modified and internally cationic PAMAM dendrimers

• QPAMAM-NHAc is a generation 4 dendrimer which is acetylated neutral surface

  • has greater cellular variability

  • reduced cyto- and genotoxicity

quantum dots (QD)

• quantum dots (QD) are very small semiconductor particles, only several nanometers in size, so small that their optical and electronic properties differ from thse of larger particles

• many types of QD will emit light of specific frequencies if electricity or light is applied to them and these frequencies can be precisely tuned by changing the dots’ size, shape and material, giving rise to many applications

• colloidal quantum dots irradiated with UV light; different sized quantum dots emit different color light due to quantum confinement

quantum dots nanocrystals

• structure of colloidal quantum dots nanocrystal

  • 15-20 nm wide

  • from inside to outside

    • core (quantum dot) → shell → polymer coating → biomolecule (antibody, etc)

• tuneability of colloidal quantum dots nanocrystals

  • 5 different nanocrystal solutions are shown excited with the same long-wavelength UV length → size of nanocrystal determines the color

    • largest to smallest: red → orange → yellow → green → blue

superparamagnetic iron oxide (SPIO) nanoparticles

• SPIO nanoparticles have been used extensively as contrast agents for MRIs

• SPIO nanoparticles should also allow the targeting of attached vectors to desired locations by the application of magnetic fields

• SPIO nanoparticles have shown promise as miniaturized heaters capable of killing cancer cells

expression of CD44 protein in ascitic cells obtained from pts with metastatic ovarian carcinoma

• CD44 antigen, cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration = one of the major cell surface markers for cancer stem cells

• cancer stem cells/cancer initiating cells = with the CD44 marker

• regular cancer cells do NOT have the marker

cancer stem cells

• cancer stem cells are the seeds of cells; primarily responsible for the initiation, extensive proliferation of the primary tumor and spreading of metastases

• stem cell characteristics

  • self-renewal

  • extensive cell proliferation

  • capability to initiate tumor growth and metastases

• CD44 antigen, a cell surface glycoprotein involved in cell-cell interactions, cell adhesion and migration is considered as one of the major cell surface markers for cancer stem cells (esp ovarian cancer stem cells)

cancer stem cells: the seeds of metastases

• important feature of cancer stem cells = ability to initiate tumor grows and metastases

• as few as 200 cancer cells displaying CD44+ phenotype form tumors in immunodeficient mice whereas 20,000 cancer that do NOT display this marker FAIL to form tumors

distribution of non-targeted and LHRH-receptor-targeted dendrimers in mice bearing xenografts of human ovarian carcinoma

• tumor cells over-expresses LHRH receptors → attaching a LHRH peptide to the dendrimer specifically guides it to the tumor

apoptosis induction and tumor size

• apoptosis and tumor volume in mice bearing xenografts of human cancer cells isolated from malignant ascites from pts with advanced ovarian carcinoma

• mice were treated 8 times 2x per week within 4 weeks starting from day 0 with indicated formulations

• WILL ASK ABOUT THIS

  • suppression of CD44 protein in cancer cells enhances anti-tumor effect of anticancer drug → limits tumor growth and prevents adverse side effects of chemotherapy

intracellular accumulation of doxorubicin

• drug sensitive cells (DOX)

  • 85% of drug stays inside the cell

  • 15% in medium

• drug resistant cells (DOX)

  • 83% of drug in medium

  • 17% in cells

• drug resistance cells (lip-DOX-MDR1 ASO)

  • 69% in cells

  • 31% in medium

  • suppression of P-glycoprotein increases drug accumulation inside cancer cells and suppresses drug resistance

personalized medicine

• often called the right treatment for the right pt and the right time

• can be used to learn about a person’s genetic makeup and to unravel the biology of their tumor; using this info to identify prevention, screening and treatment strategies that may be more effective and cause fewer side effects than would be expected with standard treatments

• plan of attack

  • analyze pt’s individual profile

  • anticipate pt reaction to certain drugs

  • overcome potential complications or adverse side effects

challenges of drug delivery for treatment of lung diseases

• limitations of systemic lung delivery

  • enzymatic degradation in the GI tract and liver

  • short half life and degradation of drugs in blood stream

  • low accumulation and retention of drugs in lungs

  • low efficacy of treatment

  • possible adverse side effects on other organs

• advantages of local inhalation drug delivery directly to the lugs

  • enhanced accumulation and retention of drugs in the lungs

  • prevention (or at least limitation) of penetration of drugs into the bloodstream and accumulation in other healthy organs

  • high efficacy of treatment

• challenge

  • majority of free drugs, native nucleic acids, and peptides CANNOT be delivered into the lungs by inhalation necessitating a special dosage form or delivery system that can be inhaled

nanotechnology based approach for treatment and imaging

• nanostructure lipid carriers (NLC)

  • carry drug and siRNA attached by disulfide bonds

  • targeting agents are attached to guide the carriers → tumors

  • PEG is used to stabilize the particles and prevent aggregation

• cationic liposomes

  • positively charged liposome with siRNA molecules attached

  • positive charge helps bind and deliver siRNA into negatively charged cell membranes

• neutral PEGylated liposomes

  • liposomes made neutral with PEGylation

  • carriers multiple things

    • CIS and DOX = chemotherapy drugs

    • MDR1 ASO = antisense oligonucleotide to block drug resistance gene

    • BCL2 ASO = antisense to block anti-apoptosis gene

• drugs

  • anticancer

  • anti=inflammatory

  • antioxidant

• nucleic acids

  • siRNA or ASO

  • targeted proteins are selected from the following classes

    • plasminogen and plasminogen activator

    • matrix metalloproteases

    • angiotensinogen

    • members of TGF-beta signaling pathways

    • chemokines

    • gamma interferon

    • interleukins and their receptors

    • tumor necrosis factor and its ligand

    • integrin

    • transforming growth factor

    • hypoxia inducible factors

    • proteins responsible for drug resistance

• targeting agent

  • LHRH for cancer

• DSPE-PEG-poly(ethylene glycol)

  • molecules were introduced to NLC in order to stabilize them against aggregation

• DOTAP

  • NLC were prepared through the incorporation of DOTAP into nanoparticle structure

organ distribution and retention of lipid nanoparticle after IV or inhalation delivery

organ distribution of NLC

• IV injection

  • 52% liver

  • 24% lungs

• inhalation

  • 75% lungs

  • 15% liver

• inhalation delivery of lipid nanoparticles leads to their preferential accumulation in the kungs

different types of vaccine

type of vaccine = virus-like particle

• licensed vaccines using this tech = HPV

• first introduced = 1986 (hep B)

type of vaccine = nucleic acid vaccine

• licensed vaccines using this tech = SARS-CoV-2

• first introduced = 2020 (SARS-CoV-2)

human coronavirus

• spike proteins found on the outside of virus

• genome contains single stranded positive-sense RNA encapsulated within a membrane with avg diameter of 75-150 nm

• envelope is covered with glycoprotein spikes giving covid their crown-like appearance

corona virus replication

• replication of COVID-19 requires a host cell and normally includes attachment, penetration, uncoating, replication, assembly and release steps

• spice glycoprotein on the surface of COVID-19 binds to angiotensin-converting enzyme 2 (ACE2) receptor protein located on the host cell plasma membrane and facilitates the host cell invasion

• KNOW HOW COVID GETS INTO CELL

  • binding and viral entry via receptor mediated endocytosis

proposed therapeutic treatments

• 2 distinct approaches are being explored for repurposing of conventional drugs and development of novel therapeutic drugs

  • prevention of virus entry into host cells

    • target ACE2 receptors and make it so that the virus CANNOT bind and infection is prevented

    • target RBD of spike protein → make things bind to the covid spikes so it can’t bind to ACE2 receptors

  • suppression of various steps in virus replication inside the cells

    • shit ton of things you can use to block steps

2 major types of COVID-19 vaccine

1. mRNA encoding the SARS-CoV spike (S) protein encapsulated in lipid nanoparticles

   1. contains mRNA instructions for making the S protein of the virus → mRNA is protected inside lipid nanoparticles → once inside your body, cells use mRNA to make S protein

2. adenovirus (AdV) vectors encoding the S protein

   1. use harmless virus (adenovirus) that carriers DNA instructions for S protein → adenovirus delivers DNA into your cells which then makes the S protein

• both gain entry into dendritic cells at the injection site resulting in production of high levels of S protein

• how immune system responds

  • dendritic cells pick up the S protein → dendritic cells release inflammatory molecules to signal danger → dendritic cells show the S protein to T cells which activate and either kill infected cells or help other immune cells → helper T cells activate B cells which turn into plasma cells that make antibodies against S protein

mRNA based COVID vaccines

• platform = lipid nanoparticle-encapsulated mRNA encoding COVID-19 S protein

• lipid nanoparticles (LPNs) are prepared through rapid mixing, often facilitated by microfluidic devices

• LPNs are formed through a cascade of merging smaller lipid vesicles

summary: strategy

• main strategies for RNA based vaccines

  • RNA type and delivery system

• 2 types of RNA

  • mRNA → basic form of RNA that instructs cells to make viral spike protein

  • self amplifying RNA (saRNA) → special type of RNA that NOT only gives the instructions but also copies itself inside the cell

• 2 main delivery systems

  • lipid nanoparticles (LPNs) → tiny fat bubbles that protect RNA and helps it enter cells

  • cationic nanoemulsions (CNEs) → positively charged emulsions that can also deliver RNA into cells (cationic helps them stick to negatively charged cell memb.)