Lecture 15: Polymer Nanoparticles

Nanoscale Analysis - Overview

• Accurately measuring analytes within living cells in real time is challenging as:

• Resolution can be lost due to sensor miniaturisation

• Components within the cell (eg proteins) can interfere with measurements

fluorophores

Individual molecular probes (free fluorescent sensing dyes) are physically small enough but usually suffer from chemical interference between probe and cellular components

Fibre-Optic Precursor Technology

• Pulled (tapered) fibre optic probes as intracellular sensors

• Tip of optical fibres coated with layer of fluorophores or the sensor material was photo-polymerized to the end of the fibre

• this probe is inserted through cell membrane

disadvantaged of Fibre-Optic Precursor

• Physical size of optical fibres limited the number of probes per cell

• Disruption of cell membrane resulted in apoptosis – not ‘passive observations’

• only measures small area of cell

types of nanoparticles used as biosensors

1. The use of polyacrylamide based nanosensors for intracellular imaging and targeted drug delivery (PEBBLEs)

2. The development of conjugated polymer nanoparticles (CPN) for use in theranostics

Probes Encapsulated By Biologically Localized Embedding (PEBBLEs)

• Based on the entrapment of fluorophores in an inert polymer matrix

• Matrix protects sensor from cellular matrix (proteins) giving a false +ve and allows cytotoxic reporter molecules to be used

• 20-200nm radius (1 ppb of cell vol.)

• Cell Viability 97% - minimal cell toxicity

• Response Time < 1 ms

• H+, Ca2+, K+, Na+, Mg2+,Cl-, NO2, O2, NO, Glucose

improving PEBBLEs

• Indirect detection: if no fluorophore for interested analyte you can embed enzymes into nanoparticle to break down analyte to a product that can be detected

• Functionalise surface: can bind Ab on surface to direct to certain parts of cell, localise detection

Manufacturing Materials

• PEBBLE polymer structure analogous to ball of rubber bands

• Three different matrices are commonly used depending on the sensor materials

  • Acrylamide

  • Liquid Polymer

  • Sol-Gel

PEBBLEs problems

Variations in fluorescence between cells may be caused by:

• Uneven fluorescent dye loading?

• Fluorophore reacting with analyte of interest?

PEBBLEs are Ratiometric Device

• As a first example of a PEBBLE sensor, we take a ratiometric oxygen sensor.

• This has a Sol Gel matrix (silica glass) and two different dyes embedded in its core.

• The Ruthenium dye (orange) fluorescence is quenched very efficiently in the presence of oxygen, so its intensity goes down with greater local oxygen concentration.

• The Oregon Green dye (green) fluorescence is not sensitive to oxygen, so its intensity stays the same and can be used as a reference.

Response & Interference

• Oregon Green (OG) can be seen to stay constant

• Ruthenium dye (RO) changes its intensity in response to varying amounts of oxygen

• Each of these peaks separately changes in intensity due to varying amounts of PEBBLEs present in the solution, or due to changes in the intensity of the excitation light.

• But, the ratio of the two peaks does not change with either of these parameters.

• So, because each PEBBLE contains both dyes, an internal reference for changes in excitation or PEBBLE concentration is always carried along with each of these nano-devices.

PEBBLEs combat interference

• serious problem of free dyes = interference by things like non-specific protein binding

• PEBBLE pores too small for proteins to get to fluorophore

• This plot shows that the Ru/Oregon Green fluorescence ratio goes up with increasing amounts of Bovine Serum Albumin (BSA) present in the solution

• The PEBBLE response stays constant - which is the desired result since the amount of oxygen in solution does not change

Quantitative Imaging

• Oxygen-sensing PEBBLEs have been used for ratiometric fluorescence imaging in rat C6 glioma cells

• Computer software used to superimpose the images, showing where signals are coming from within the cells.

• Changes in the fluorescence used to show local changes in oxygen concentration

• ratio images can be generated showing oxygen concentration based on test-tube calibrations of the sensors.

Delivery Methodology: Gene gun

• originally to deliver DNA into cells, but can also be used for PEBBLEs.

• blast of helium shoots dried PEBBLEs off a disk, are propelled down into the cell culture dish and embedded randomly

• Gene gun forces nanosensors through the cell membrane

• Nanosensors also breach the nuclear membrane – possible trigger of cell death

• Not ‘passive observation’

Delivery Methodology: Cellular internalisation of nanosensors

• For phagocytic cells such as macrophages and glial cells

• Sensors ingested via routine cell process

• Remain encapsulated within phagosome

• Nanosensors are not free in cell cytoplasm

• initially used to detect free radicals

Delivery Methodology: liposomal transfection

• for non-phagocytic cells (eg glioblastoma)

• probably best

• No natural endocytic pathways

• Exploit transfection reagent for liposomal cargo delivery

• Nanosensors are free within the cytoplasm

• Check internalisation with confocal microscopy

• minimal disruption of cellular processes

why is PEBBLE great?

• Some nanotechnology production techniques require lots of time and expertise to accomplish.

• In contrast, PEBBLE production is rooted in basic chemistry methods.

• This technology is quite cheap, easy and fast.

How to make polyacrylamide PEBBLEs

1. Water in oil microemulsions are used for the radical mediated polymerisation

2. dissolve polymers in organic solvent

3. add fluorophore

4. evaporate solvent

5. The PEBBLEs are then concentrated

6. washed and vacuum filtered

7. The resulting powdered nanosensors can be stored indefinitely at RT before resuspension and use in sensor work

Surface Modification

• involves polymerization of the PEBBLEs using a mixture of monomers, some having free amine groups

• Some of these groups will be exposed on the outside of the PEBBLE, and simple attachment can be made to other molecules (like biotin) through carboxyl or succinimydal ester groups

• can be used to attach Ab

The development and in vitro characterisation of an intracellular nanosensor responsive to reactive oxygen

• Reactive oxygen species (O2-, NO and H2O2) play an important role in many pathological processes (eg stroke, arthritis, skin ageing)

• Quantification of ROS is difficult due to their innate reactivity

• Nano sensor technology could offer a unique insight into ROS-mediated intracellular events

• Xanthine oxidase produces ROS

• increase in enzyme = increase in fluorescence

• NR8383 mouse alveolar macrophage cells

  • engulfed NP in phagosomes

  • overlaying images can identify leaks

  • stimulate free radical response

  • can measure free radical production in real time

Enhanced uptake of nanoparticle drug carriers via a thermoresponsive shell enhances cytotoxicity in a cancer cell line

• standard chemotherapy drugs are predominatindly hydrophobic

• need large doses= side effects

• Tumours are slightly hyperthermic (+1–2 °C) compared to surrounding healthy tissue

• Encapsulate drug in a ‘thermoresponsive vehicle’ to target the therapy to tissue at elevated temperature while leaving other tissues unaffected

• Hyperthermic region can be defined by the application of external ultrasound, or by near-infra-red irradiation

• outside capsule is temperature sensing hydrophobic polymer strands

• increase temp to 40c > hydrophobic chains collapse > can be absorbed by tumour cell

Cytotoxicity of Paclitaxel (PTX)-loaded nanoparticles in Breast Cancer (MCF-7) cells

• Paclitaxel used to treat breast cancer

• Cells were incubated with either free PTX or nanoparticles containing PTX at normal physiological (37 °C) or mildly hyperthermic (40 °C) temperature.

• Cellular metabolic activity was determined using an MTT assay

• Free PTX displayed the same effect on metabolic activity at both temperatures indicating that temperature variation over the 37–40 °C range did not affect uptake of the free drug or its inherent efficacy.

• At 37 °C, nanoparticles containing PTX displayed toxicity comparable to, or lower than, that of free PTX – reduced nanoparticle uptake of PTX?

• Conversely, at 40 °C toxicity of PTX nanoparticles was greater than that of free PTX most likely due to increased particle uptake and release of PTX within the cell.

Conjugated Polymer Nanoparticle (CPN®)

• Excellent stability – 20 months, pH 2 – pH 10, up to 120°C

• Unaffected by photobleaching in cells and samples

• Non-toxic – heavy-metal free, organic

• Contains iron oxide – for magnetic manipulation

• VERY fluorescent

• Fluorescence emission wavelengths from 420 nm to 1130 nm

• Familiar surface ligand chemistry – for attaching targeting biomolecules (Ab, oligonucleotides, streptavidin, etc.)

• Storage at ambient temperatures – unless linked to a biological molecule, then store at 4°C

Theranostic NIR-active conjugated polymer nanoparticles

• Three conjugated polymer nanoparticles (CPNs) were developed using PTB7, a material commonly used in solar cells.

• The CPNs show strong absorption and emission in the NIR-I window (650–950 nm), ideal for biological imaging.

• NIR imaging capability was demonstrated in A549 human lung epithelial cells.

• The CPNs generate reactive oxygen species (ROS) upon UV photoexcitation, confirmed in aqueous solutions and HaCaT cells.

• This ROS generation leads to phototoxic effects, useful for therapeutic purposes.

• PTB7’s dual functionality—NIR imaging and ROS production—makes it suitable for theranostic applications.

• The system offers potential for photodynamic therapy (PDT), enabling both cancer detection and treatment.

• PTB7’s prior use in solar energy and display technology stems from its NIR optical properties, which are also advantageous in biomedical applications.

Photodynamic Therapy (PDT)

• PDT relies on a photo-induced generation of reactive oxygen species by a photosensitizer (PS)

• Used clinically in the treatment of acne, precancerous cells and sun-damaged skin

• PDT Advantages:

  • Minimal invasiveness or damage to healthy tissues

  • Lack of drug resistance

  • Applicable to a broad spectrum of cancer targets

• can bind NP to specific Ab for tumour Ag, irradiate with light and cause free radical production to kill cell

Key Photosensitiser Properties

• Biocompatible

• Excellent ROS generation ability

• Strong near-infrared (NIR) absorption

• NIR = longer WL = will pass through skin to tumour

Production of CPNs using Nanoprecipitation

1. polymer strands in stir in pot of water with co polymer

2. CPNs co-precipitated with a copolymer

Optical properties of PTB7-based CPNs in aqueous solutions.

• co-polymer effects maximum fluorescence

• may be due to polymer degradation, how well does it withstand free radical reduction

CPN Dependent Superoxide production in response to photostimulation at 365nm

1. DTSSP on surface of gold electrode, banded to cytochrome c

2. Superoxide (O₂⁻) reducing the Fe³⁺ in cytochrome c to Fe²⁺

3. electron taken by +ve electrode surface

4. process repeated by present free radicals