l3: nanomedicine in detoxification course 207

L3: Nanomedicine in Detoxification

April 10, 2018

NANO 243/CENG 207 Course Use Only

Current and Forthcoming Approaches for

Systemic Detoxification

Theme Editors:

Liangfang Zhang & Jean-Christophe Leroux

“At first sight, it may appear a bit contradictory to dedicate a special issue of Advanced

Drug Delivery Reviews to technologies aimed at removing, rather than delivering,

compounds from the body. ”

“Upon closer inspection, the commonalities between these two opposing fields become

more apparent, as many new approaches in the field of detoxification rely on established

drug delivery systems, including different forms of nanoparticle technology.”


▪ Intravenous fat emulsions (IFE, e.g., liposomes) to treat drug overdose

▪ Enzymatic nanoparticles for organophosphates

▪ Enzyme-nanocarriers for systemic detoxification

▪ Molecular imprinted polymer (MIP) as antidots

▪ Nanosponge technology as a broad-spectrum detoxification system

▪ Actively-moving detoxification system

Detoxification Systems


(1) Emulsions and Liposomes to Treat Drug Overdose

Over one million cases of drug intoxication are reported yearly in USA. Rationale behind the use of transmembrane pH-gradient liposomes as antidotes. Similarly to IFE, the vesicles act as sinks to scavenge circulating free drugs. The transmembrane pH gradient maximizes the quantities that can be captured. Sequestration of the drug by vesicles in the blood decreases the amounts of pharmacologically active free drug.


DTZ uptake in HBS and in the presence of 50% plasma by different colloidal formulations, including the commercially available IFE Intralipid. Transmembrane pH-gradient liposomes surpassed the IFE (Intralipid 20%) by 40-fold in terms of capture capacity. They were also largely superior to neutral liposomes with an internal pH of 7.4 and negatively charged liposomes (prepared with anionic DMPG instead of Egg PC).

pH-Gradient Liposomes


Mechanisms of Liposomal Detoxification

Plausible locations for two different

classifications of drug to bind to anionic-

charged liposomes. (A) shows the

potential binding site of a tricyclic

antidepressant (TCA) where the

uncharged portion of the drug aligns

within the lipid bilayer and the positively

charged head groups align with the

negatively charged phospholipid head and

aqueous bulk. (B) shows the potential

binding site of a local anesthetic where

two uncharged portions align within the

lipid bilayer and positively charged groups

interact with the negatively charged phospholipids.

Adv. Drug Deliv. Rev. 2015, 90, 12–23.


(2) Enzymatic Nanoparticles for Organophosphate

Schematic of RBC-NPs (Nanosponges) as anti-OP bioscavengers for treating OP poisoning. With no

treatment (left), dichlorvos (DDVP), a model OP, irreversibly binds acetylcholinesterase (AChE), preventing

the breakdown of acetylcholine (ACh) into choline and acetate. When RBC-NPs are introduced (right), they

scavenge free DDVP molecules in circulation, preserving the ability of endogenous AChE at the synapse to

perform the function of breaking down ACh.NANO 243/CENG 207 Course Use Only

In Vivo Neutralization of DDVP

In vivo neutralization of DDVP by RBC-NPs. (A) Survival curve of mice over 16 days and (B) relative RBC

AChE activity of mice following intravenous administration of 200 mg/kg of RBC-NPs or PLGA-PEG NPs

immediately after an intravenous injection of DDVP at a lethal dose (10 mg/kg) (n = 10). (C) Survival curve of

mice over 16 days and (D) relative RBC AChE activity of mice following administration of 200 mg/kg of RBC-

NPs or PLGA-PEG NPs immediately after oral administration of DDVP at a lethal dose (150 mg/kg) (n = 10). NANO 243/CENG 207 Course Use Only

RBC AChE Activity Recovery

RBC AChE activity recovery following OP detoxification by RBC-NPs. (A,B) Relative RBC AChE

activity recovered over a span of 4 days after the mice were challenged (A) intravenously with DDVP (10

mg/kg) or (B) orally with DDVP (150 mg/kg), and immediately treated with RBC-NPs (200 mg/kg) (n=10).

(C) Biodistribution of RBC-NP/DDVP complex 24 h after intravenous injection. (D) Hematoxylin and eosin

(H&E) stained liver histology showed no tissue damage on day 3 (top) and day 7 (bottom) following RBC-

NP/DDVP complex injections.

ACS Nano 2015, 9, 6450-6458


(3) Enzyme-Nanocarriers for Detoxification

A schematic illustration of the three major types of enzyme-nanocarrier architectures for

detoxification through intravenous delivery.NANO 243/CENG 207 Course Use Only

Enzyme-Loaded Liposomes

A schematic illustration of preparing liposomes and stealth liposomes through self-assembly

of lipids or a mixture of lipids and PEG-lipids, respectively. Stealth liposomes could also be

prepared by conjugating PEG chains onto the liposome surface.


PEGylated Enzymes

A schematic illustration of a PEGylated protein. The PEG chains are conjugated on an

enzyme, which could protect the protein from proteolysis and help to evade the immune

system.NANO 243/CENG 207 Course Use Only

Enzyme-Loaded Nanocapsules

Schematic representing the preparation of single enzyme nanocapsule via in situ synthesis of

crosslinked polymer shell on the enzyme surface. Enzyme nanocapsule is synthesized by

firstly conjugating or adsorbing unsaturated molecules onto the protein surface, followed by

the initiation of in situ polymerization reaction to form the polymer shell.

Adv. Drug Deliv. Rev. 2015, 90, 24–39.


(4) Molecularly Imprinted Polymer (MIP) as Antidotes

A Molecularly Imprinted Polymer (MIP) is a polymer that has been processed using

the molecular imprinting technique which leaves cavities in polymer matrix with affinity

to a chosen "template" molecule. The process usually involves initiating the

polymerization of monomers in the presence of a template molecule that is extracted

afterwards, thus leaving complementary cavities behind. These polymers have affinity

for the original molecule and have been used in applications such as chemical

separations, catalysis, or molecular sensors.


One MIP Example

(a) Monomers used for NP synthesis.

(b) Amino acid sequence of melittin.

(c) Schematic representation of the

melittin imprinting process.

Hydrophobic, positive/negative

charged, and hydrophilic residues are

printed in brown, blue/red, and green.

J. Am. Chem. Soc., 2008, 130, 15242-15243


Screening of Effective MIP Formulations

Inhibition of hemolytic activity of melittin by polymer NPs incubation at 37 °C without melittin (tube 1), with 1.8 μM melittin (tube 2), with 1.8 μM melittin and 0.12 mg/ml NPs (1-7)


Validation of MIP Detoxification In Vivo

Survival curves of mice over 24 h after injection (IV) of 4.5 mg/kg melittin (green) with 30 mg/kgMIP NPs (red), or non-imprinted NPs (gray).NANO 243/CENG 207 Course Use Only

Technology: The nanopsonge is a biocompatible particle made of a polymer core wrapped

in a red blood cell membrane. It is capable of safely removing a broad class of dangerous

toxins from the bloodstream regardless of the toxin’s molecular structure.

Caption:

Left: Schematic illustration

Right: TEM images

(5) Nanosponge Technology


Bacterial Toxin Neutralization

RBCs + α-toxin

0 4 8 12 16 20 24 3600

20

40

60

80

100

No treatment

PEG-PLGA nanoparticle

RBC vesicle

Nanosponge

Time after injection (hr)

Su

rviv

al r

ate

(%

)

RBCs + α-toxin

& nanosponge

Hemolytic Assay: Centrifuged RBCs after incubation with

α-toxin mixed in PBS or nanosponges.

In vivo Toxin Neutralization: 72 hr after the s.c.

injection of 1.8 μg of free α-toxin, severe skin

lesions were induced. 100 μg of the nanosponges

(toxin-to-nanosponge ratio ≈ 70:1) appeared to

neutralize the toxin.

In vivo Detoxification: Mice without any treatments had a

100% mortality rate within 6 hr following a bolus lethal dose of

α-toxin (75 μg/kg). In contrast, nanosponge pre-inoculation (80

mg/kg ), reduced the mortality rate markedly to 11% (n=9).

α-toxin α-toxin + nanosponge


Live

r

Kidne

y

Splee

nLu

ng

Hea

rt

Blood

0

20

40

60

80

100

Na

no

sp

on

ge

/a-to

xin

dis

trib

utio

n (%

)

Biodistribution

Fate of Sequestered Toxins

Liver histology

Day 3 Day 7

Nature Nanotech., 2013, 8, 336-340.


10-1 100 1010.0

0.2

0.4

0.6

0.8

1.0

Control

Nanosponge (500 mg/mL)

Nanosponges Neutralize Multiple Toxins

α-toxin (μg/mL) Streptolysin-o (μg/mL) Melittin(μg/mL)

Rela

tive V

iabili

ty

Rela

tive V

iabili

ty

Rela

tive V

iabili

tyNANO 243/CENG 207 Course Use Only

(6) Actively-Moving Micromotors for Detoxification

(A) Schematic preparation of motor sponges

(ultrasound-propelled gold nanowire motor

coated with RBC membranes).

(B) SEM image of a fabricated motor sponge.

(C) Fluorescent image of a motor sponge.

(D) Determination of the membrane coverage

on the motor sponges.

(E) Evaluation of the orientation of RBC

membranes using glycoprotein assay.


Ultrasound Propulsion of Motor Sponges

(A) Schematic illustration of ultrasound-propelled motor sponges.

(B) Time-lapse images illustrating random motion and moving distances of three motor sponges.

(C) A large population of moving motor sponges under the ultrasound field.

(D) Fluorescence spectra illustrating the stability of RBC membrane coating on the motor sponges.

(E) Speed of motor sponges (red line) and bare motors (black line) upon the ultrasound voltage.

(F) Speed modulation of motor sponges in response to a Low/High/Low (1/3/1 V) 6 s potential steps. NANO 243/CENG 207 Course Use Only

Propulsion of Motor Sponges in Whole Blood

(A,B) Time-lapse images showing the propulsion of motor sponges in the whole blood over 40 minutes.

(C,D) Time lapse images showing the propulsion of motor sponges in whole blood before and after 48 h

incubation in the whole blood, respectively.


In Vitro Neutralization of Toxins by Motor Sponges

(A) Schematic mechanism for the neutralization of mellitin toxin using motor sponges.

(B) Centrifuged RBCs after incubation with melittin mixed different treatment groups.

(C) Hemolysis analysis of samples in (B) as the function of time (0-30 min).

(D) Percentage of hemolysis of samples in (B) after 10 min incubation.

Advanced Functional Materials 2015, 25, 3881-3887.


l3: nanomedicine in detoxification course 207

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