November 07, 2011

Kristi Hemstreet

Journal of Inorganic Biochemistry, Vol. 103, 2009, p. 1039-1044

Ferritin is a 450 kDa globular protein complex composed of 24 subunits

The primary intracellular iron-storage protein in prokaryotes and eukaryotes

Each molecule of ferritin can store approximately 4500 iron (Fe3+) ions

Dimensions of the ferritin/apoferritin protein cage:12-nm diameter with a 8-nm hollow interior

Channels are formed at the intersections of the subunits - allow the transport of certain ions and molecules

In vertebrates, ferritin consists of both Light (19 kDa) and Heavy (21 kDa) type subunits

4-helix bundle structure connected by loops

The ferritin H subunit possesses ferroxidase activity - catalyzes the oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+)

The ferritin L subunit plays a role in iron nucleation and protein stability

Once inside the ferritin shell, iron ions form crystallites with phosphate and hydroxide ions

Light polypeptide

Heavy polypeptide

Iron is essential for normal cell growth and proliferation, necessary for oxygen transport in the blood

Excess iron is toxic to cells - formation of damaging reactive oxygen species via the Fenton Reaction

Apoferritin binds to free ferrous iron and stores it in a non-toxic, soluble form

Iron can be extracted from ferritin when needed by the reticuloendothelial system

Ferritin serves as a buffer against iron deficiency and iron overload.

Normal blood levels of iron range from 30-300 ng/ml in males and 15-200 ng/ml in females

Ferritin test – serum ferritin levels are measured to determine the total amount iron stored in the body

Insufficient iron levels (<50 ng/ml) can indicate anemia, hypothyroidism, vitamin C deficiency, celiac disease, and restless legs syndrome

Excess iron levels can indicate inflammation, liver disease, chronic infection, autoimmune disorders, hemochromatosis, multiple blood transfusions, and some types of cancer

Protein-based nanomedicine platforms are ideal for drug delivery – biocompatibility, biodegradability, low toxicity, uniform size, multifunctionality

The ferritin/apoferritin protein cage is a protein nano-particle that has been used and characterized for drug-delivery systems (DDS)

Passable hydrophobic and hydrophilic channels through the cage - containment of both insoluble and soluble drugs for therapeutic and imaging purposes

Ability to disassemble and reassemble under pH control allows the loading of numerous potentially therapeutic compounds

The use of platinum-based anticancer drugs is limited due to severe general toxicity and drug resistance

Conventional or free drugs do not act in a localized way – widely distributed

Free drug is quickly eliminated from the blood by the kidneys, or through immune recognition

It can take 1-3 days for an anticancer agent to effectively infiltrate the tumor cells

How can we accelerate the uptake of the drug? QQ protein delivery!

Utilizes the accumulation of nanomedicine platforms into tumor interstitial fluid

Long systemic circulation times

Increased vascular permeability and increased retention

Enhanced drug delivery to the tumor site due to the EPR (enhanced permeability and retention) effect

Less normal tissue damage

Ruimin Xing, Xiaoyong Wang, Chanli Zhang, Yangmiao Zhang, Qi Wang, Zhen Yang, Zijian Guo

Nanjing University, Nanjing, PR China

Journal of Inorganic Biochemistry, Vol. 103, 2009, p. 1039-1044

Purpose: to describe an alternative strategy for the delivery of platinum drugs (cisplatin, carboplatin, and oxaliplatin) by encapsulation in the cavity of apoferritin

Drug delivery systems with tumor-targeting potential are highly desired for improving the efficacy and applicability of platinum-based anticancer drugs

From the group’s recent work, AFt has been shown to be a promising vehicle for targeted delivery of platinum-based drugs

Water soluble Least water soluble

AFt was obtained from horse spleen ferritin by demineralization.

The platinum drugs were dissolved, respectively, to get their saturated solutions (CDDP, 1 mg/ml; CBDCA, 15 mg/ml; LOHP, 5 mg/ml).

AFt was added to each to reach a final protein concentration of 1 mg/ml.

Scheme 1. Schematic illustration of the pH-mediated encapsulation of cisplatin (CDDP), carboplatin (CBDCA), or oxaliplatin (LOHP) by apoferritin (AFt) via an unfolding-refolding process.

AFt and AFt-drug complexes were characterized by: 1) UV-vis spectrometry – to examine whether the structure of AFt has been changed

2) Circular dichroism spectrometry – to determine any possible changes in secondary structure of AFt

3) Dynamic light scattering – determine the hydrodynamic diameters of AFt and AFt-drug complexes

4) Zeta potential – an indication of surface charges on a particulate species

5) Pt analysis – ICP-MS and BCA assay

6) Cytotoxic assay – antitumor potential was tested against rat pheochromocytoma cell line (PC12)

7) Cellular uptake – the uptake of Pt in PC12 cells was determined by ICP-MS

Fig. 3 - The hydrodynamic diameters (Φ) determined by DLS are in agreement with the previous report. The size differences among AFt and AFt-drug complexes are negligible, suggesting AFt is still properly assembled after the encapsulation of drugs.

Table 1 – Zeta potential of AFt and AFt-drug complexes with respective experimental errors. The electrostatic property on the surface of AFt hardly changed after platinum drug encapsulation, binding affinity should remain unchanged.

AFt was used as a carrier to encapsulate three platinum anti-cancer drugs, CDDP, CBDCA and LOHP, which resulted in some tumor targeting property

The structural integrity of AFt is well preserved in the AFt-drug complexes

AFt-CDDP has an increase in cellular uptake as compared with CDDP

AFt-CDDP may be able to defeat CDDP-related drug resistance

AFt has tumor targeting potential, relative high loading capacity and good water solubility, which make it a promising carrier for Pt-based anticancer drugs

QQ-protein transduction technology - Intracellular Protein Therapy QQ-protein transduction technology - Intracellular Protein Therapy

We also applied ferritin system for brain cancer detection.

Glioblastoma multiforme (GBM) is the most common and most aggressive type of primary brain tumor in humans.

Despite being the most prevalent form of primary brain tumor, GBMs occur in only 2-3 cases per 100,000 people in Europe and North America.

Median survival with standard-of-care radiation and chemotherapy with temozolomide is 15 months. Median survival without treatment is 4 1/2 months.

Early detection is critical for prolonged median survival of GBM.

Although MRI was initially hoped to provide a means of making definitive diagnoses Although MRI was initially hoped to provide a means of making definitive diagnoses

noninvasively, it is found that the addition of contrast agents in many cases improves noninvasively, it is found that the addition of contrast agents in many cases improves

the sensitivity and/or specificity. Gadolinium (Gd) is used as a positive contrasting the sensitivity and/or specificity. Gadolinium (Gd) is used as a positive contrasting

reagent, giving a brighter imaging of the tumor.reagent, giving a brighter imaging of the tumor.

MRI Detection of GlioblastomaMRI Detection of Glioblastoma

We want to use MRI imaging to detect glioma

In order to clearly detect glioma using MRI we need a contrasting reagent

Iron serves as a good negative contrasting reagent – often rich in neoplastic and nerve cells

As I illustrated to you, ferritin can hold up to 4500 iron ions

Ferritin is also a protein nanoparticle and therefore utilizes the EPR effect for targeted delivery of iron to the tumor

This work was done in collaboration with Dr. Feng Jiang and Dr. Xuguang Zheng at Henry Ford Health System.

The protocol for the experiment is shown above.

9L cell line: a rat glioma cell line.

9L-injection

Ferritin only iv injection QQ-ferritin iv injection

QQ modification is necessary for ferritin to penetrate the tumor cells

Prussian blue stains for iron

Immunostaining of glioma brain tissue slides using anti-Ferritin Ab and anti-actin Ab

Tumorcenter

Tumorcenter

Tumorcenter

Ferritin is co-localized in the capillary blood vessel

Ferritin is located inside glioma cells

Zoomed-in view Zoomed-in viewFerritin is co-localized in the capillary blood vessel

Ferritin is located inside glioma cells

Ferritin: RedVessel: GreenMerge: YellowBlue: nuclei (DAPI)

Ferritin: RedVessel: GreenMerge: YellowBlue: nuclei (DAPI)

Ferritin: RedVessel: GreenMerge: YellowBlue: nuclei (DAPI)

Ferritin: RedVessel: GreenMerge: YellowBlue: nuclei (DAPI)

Using QQ-ferritin, we can perform detection of glioma in the rat brains with enhanced conntrasting, possibly for early glioma detection.

Since the ferritin cage is large and can load chemo-therapeutic drugs such ascarboplatin, we would like to follow the paper that I presented to load carboplatininto ferritin for early detection of glioma and for glioma treatment.

We started this work a few months ago, and have some data to present.

Left: 8% Native gel – Ft before and after encapsulation with CBDCA1) Ft (38.5 μg/ml)2) Ft-CBDCA 6.5x dilution

8% Native gel – working stock Ft (40μg/ml) compared with Ft-CBDCA

1) Ft (40μg/ml)2) Ft-CBDCA 8.3x dilution3) Ft (40μg/ml)4) Ft-CBDCA 5x dilution5) Ft (40μg/ml)6) Ft-CBDCA 3.1x dilution7) Ft (40μg/ml)8) Ft-CBDCA 2.5x dilution9) Ft (40μg/ml)10) Ft-CBDCA 1.7x dilution

1 2 3 4 5 6 7 8 9 10 1 2

Left: Ft before and after fluorescence labeling

1) BenchMark Protein Ladder2) Ft 5x dilution (8μg/ml)3) Ft488dialyzed 5x dilution

BCA Assay was performed using the dialyzed sample of Ft488 (5x dilution). Average concentration = 2.39mg/ml.

Right: Ft488 before and after QQ modification

1) BenchMark Protein Ladder2) Ft3) Ft488dQQ

1 2 3 1 2 3

70 kDa- 60 kDa- 50 kDa-

40 kDa-

30 kDa-

20 kDa-

60 kDa- 50 kDa-

40 kDa-

30 kDa-

20 kDa-

Wang Lab:Dr. Jianjun WangDr. Qianqian LiDr. Jianglei ChenDr. Victoria MurrayDr. Yuefei HuangCarrie O’Connor, M.S.

Collaborators, Henry Ford Health System:Dr. Feng JiangDr. Xuguang Zheng