Cancer therapy has many faces: surgery, chemotherapy, radiotherapy, targeted drugs, immunotherapy… But deep inside the cell, there is another very interesting target: telomerase.

Telomerase is an enzyme that helps cancer cells live longer than they should. Blocking telomerase is a promising way to slow down or stop tumor growth. However, even when we have a molecule that can block telomerase, we often face a very practical problem: how do we deliver it into cancer cells efficiently, especially if the drug does not dissolve in water?

This is exactly the problem the article focuses on. The researchers studied a well-known telomerase inhibitor called BIBR 1532, and they tried to deliver it using a special porous material called ZIF-8, which belongs to the metal–organic framework (MOF) family.

The core idea of the paper is:

“BIBR 1532 works, but it is poorly soluble in water and doesn’t get into cells very well. ZIF-8 can act as a nano-carrier that helps transport BIBR 1532 into cancer cells, helps it escape from lysosomes, and finally increases its effect on telomerase and cancer cell growth.”

Let’s unpack this in a simple way.

Background: What Is Telomerase and Why Do We Want to Inhibit It?

At the ends of our chromosomes, we have telomeres—repetitive DNA sequences that protect our genetic material, a bit like the plastic caps at the ends of shoelaces.

Every time a normal cell divides, these telomeres get a little shorter. After many divisions, telomeres become too short, and the cell goes into senescence (a permanent “sleep” state) or dies. This is one of nature’s safety mechanisms against uncontrolled growth.

Telomerase is an enzyme complex that can extend telomeres, allowing cells to divide more times than they normally would. In most normal body cells, telomerase activity is very low or absent. In contrast, in the vast majority of human cancers, telomerase is switched on and very active. This is one reason tumors can keep growing.

The key protein component in human telomerase is called hTERT (human telomerase reverse transcriptase). If you block hTERT, you interfere with telomerase function. Over time, telomeres shorten, and cancer cells can be pushed towards senescence or death.

Because of this, telomerase and hTERT are very attractive targets for broad-spectrum anticancer therapies.

BIBR 1532: A Good Inhibitor With a Practical Problem

BIBR 1532 is a synthetic, non-nucleosidic, non-peptidic telomerase inhibitor. It binds to a conserved hydrophobic pocket (called the FVYL motif) in hTERT and blocks the enzyme’s activity. Many studies have shown that BIBR 1532 can:

• Gradually shorten telomeres

• Inhibit cancer cell proliferation

• Enhance the effects of other treatments (e.g., with paclitaxel, emodin, or radiation)

However, BIBR 1532 has a serious drawback:

• It is poorly soluble in water.

This has several consequences:

• It does not disperse well in biological fluids.

• Cellular uptake is low.

• The effective dose at the target (inside cells, near the nucleus) is limited.

• At low doses and short times, the growth inhibition is often weak.

Some groups tried to improve its performance by modifying the chemical structure of BIBR 1532 or combining it with other drugs or therapies. These strategies helped, but they did not directly solve the basic physical problem: the drug itself is hydrophobic and doesn’t dissolve well in water.

So the authors of this paper asked a different question:

Can we keep BIBR 1532 as it is, but package it inside a smart nanocarrier that improves its solubility, transport, and release inside cancer cells?

That’s where MOFs and ZIF-8 come in.

Metal–Organic Frameworks and ZIF-8: Why Are They Interesting for Drug Delivery?

Metal–organic frameworks (MOFs) are crystalline materials built from:

• Metal ions (or clusters)

• Organic ligands (like carboxylates, imidazoles, etc.)

They form 3D porous networks with:

• Very high surface area

• Tunable pore sizes

• Adjustable chemistry

• High loading capacity for guest molecules (like drugs)

This combination makes MOFs very attractive as drug carriers. They can:

• Protect sensitive molecules

• Load relatively high amounts of drug

• Release the drug in response to certain triggers (like pH)

Within the wide MOF family, ZIF-8 is especially popular for biomedical applications.

What is ZIF-8?

ZIF-8 (Zeolitic Imidazolate Framework-8):

• Is built from zinc ions and 2-methylimidazole ligands

• Forms a cage-like 3D network with micropores

• Has high surface area and a tunable porous structure

• Is stable in neutral conditions, but degrades in acidic environments

This last point is extremely important:

• Outside the cell (blood, culture medium), the pH is around 7.4 → ZIF-8 is stable.

• Inside lysosomes (acidic compartments in cells), the pH is around 5 → ZIF-8 can break down.

Because of this pH sensitivity, ZIF-8 can:

• Remain intact during circulation and uptake

• Degrade in acidic lysosomes, releasing the drug inside the cell

In addition, the imidazole ring in ZIF-8 can become protonated in acidic lysosomes. This creates a “proton sponge” effect:

• Protons (H⁺) are pumped into the lysosome.

• Imidazole groups get protonated and buffer protons.

• To balance charges, ions and water flow in.

• Lysosomes swell, the membrane permeability increases, and sometimes the membrane partially ruptures.

• This helps escape of the cargo (drug) from lysosomes into the cytoplasm.

Many previous studies already showed that ZIF-8 can protect and deliver biological macromolecules (enzymes, DNA, RNA, CRISPR/Cas9) and small drugs more efficiently, by using exactly this protonation-driven lysosomal escape.

So ZIF-8 is:

• A nanoporous container

• A pH-sensitive release system

• A lysosome escape helper

That makes it a very interesting candidate to deliver hydrophobic BIBR 1532 to cancer cells.

How Did the Authors Build and Characterize the BIBR 1532@ZIF-8 System?

Synthesis of ZIF-8

The researchers first synthesized ZIF-8 nanoparticles in water using:

• Zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O)

• 2-methylimidazole

They mixed the solutions, stirred for several hours, collected the particles by centrifugation, washed them, and dried them.

They then used several standard characterization techniques to check what they had made:

• SEM and TEM images: showed regular hexagonal / octagonal particles around 100–200 nm in size.

• Dynamic light scattering (DLS): confirmed a hydrated particle size around ~130 nm with low polydispersity, meaning the particles were reasonably uniform in solution.

• Zeta potential: about +18 mV, indicating a moderately positive surface charge.

• FT-IR: showed bands typical for 2-methylimidazole vibrations and confirmed the framework.

• XRD: matched the expected patterns for ZIF-8, confirming the correct crystal structure.

• TGA: showed that ZIF-8 was thermally stable up to a few hundred degrees before decomposing.

These results together showed that ZIF-8 was successfully synthesized with the expected structure.

Loading BIBR 1532 into ZIF-8

To load BIBR 1532, they co-synthesized BIBR 1532@ZIF-8:

• Zinc salt and BIBR 1532 were dissolved together.

• This mixture was added into a 2-methylimidazole solution under stirring.

• As ZIF-8 formed, BIBR 1532 became encapsulated inside the framework.

The resulting composite was again washed and dried.

Characterization of BIBR 1532@ZIF-8 showed:

• SEM/TEM: particles still had regular ZIF-8-like shapes (hexagonal / octagonal).

• DLS: average size increased somewhat to ~165 nm, with a slightly higher polydispersity.

• Zeta potential: became negative (around −14 mV), reflecting the presence of BIBR 1532 and changes in surface chemistry.

• FT-IR: showed the vibrational bands of ZIF-8 plus a band associated with BIBR 1532’s –OH group, indicating successful encapsulation.

• XRD: similar structure to ZIF-8, suggesting the framework was maintained.

• TGA: the weight-loss curve changed compared to pure ZIF-8, also supporting successful loading.

Importantly, they also evaluated dispersion and stability in water:

• Free BIBR 1532 precipitated quickly in water; the suspension became clear with sediment at the bottom within a day.

• BIBR 1532@ZIF-8, by contrast, stayed well dispersed in water for at least a week.

The drug loading was determined (using a fluorescently labeled version of BIBR 1532 and a standard curve) to be about 6% by weight – meaning 100 mg of BIBR 1532@ZIF-8 contained about 6 mg of BIBR 1532.

Even though this loading is not extremely high, it turned out to be enough to greatly enhance the biological effect.

Where Does ZIF-8 Go Inside Cells, and What Does It Do There?

Cytotoxicity of ZIF-8 Alone

Before using ZIF-8 as a carrier, the authors checked its toxicity in A549 lung cancer cells:

• At concentrations up to 100 µg/mL, ZIF-8 did not significantly reduce cell viability after 24 hours.

• At 150 µg/mL, a small cytotoxic effect appeared.

So within the working range, ZIF-8 itself was fairly well tolerated by the cells.

Cellular Uptake and pH Sensitivity

To see how ZIF-8 behaves inside cells, they:

• Labeled ZIF-8 with fluorescein (green fluorescent dye).

• Exposed A549 cells to fluorescein@ZIF-8.

• Tracked intracellular zinc levels using ICP-OES.

They observed:

• Cellular uptake increased rapidly within the first hour.

• After that, uptake slowed and approached saturation by about 12 hours.

They also looked at pH-dependent stability in vitro:

• In neutral solution (pH 7.4), the average particle size of ZIF-8 stayed stable over many hours.

• In acidic solution (pH 5.0, similar to lysosomes), the particle size decreased over time until it became undetectable—indicating gradual degradation.

Lysosomal Localization and Membrane Permeability

Using confocal microscopy, they co-stained:

• Fluorescein-labeled ZIF-8 – green

• Lysosomes – red (LysoTracker)

• Nuclei – blue (Hoechst)

At about 1 hour, ZIF-8 was clearly localized in lysosomes (yellow spots from overlapping green and red signals). As time passed:

• ZIF-8 signal decreased in lysosomes and cytoplasm.

• By 6 hours, the green fluorescence was almost gone, suggesting clearance/degradation.

They then asked: Does ZIF-8 change lysosomal membrane permeability?

They used acridine orange (AO), a dye that:

• Shows red fluorescence inside intact, acidic lysosomes (where it accumulates).

• Shows green fluorescence in the cytoplasm if it leaks out due to lysosomal membrane damage or permeabilization.

In control cells, the ratio of green/red fluorescence stayed stable over 24 hours. In ZIF-8-treated cells:

• Over time, the green/red ratio increased – red (in lysosomes) decreased, green (in cytoplasm) increased.

• This indicates that lysosomal membranes became more permeable, and AO leaked out.

However, when they looked at the expression of key lysosomal proteins:

• LAMP1 (lysosomal membrane protein)

• Cathepsin B (lysosomal enzyme)

they did not see major changes after ZIF-8 treatment. So ZIF-8:

• Does alter lysosomal membrane permeability, likely via the protonation (proton sponge) effect,

• But does not strongly damage or destroy lysosomes in a way that changes these marker protein levels in this time frame.

Encapsulating BIBR 1532 in ZIF-8 Changes Its Journey and Destination

To visualize BIBR 1532 inside cells, the authors:

• Labeled BIBR 1532 with 6-aminofluorescein (6-AF) (green).

• Confirmed successful labeling via FT-IR.

• Encapsulated 6-AF@BIBR 1532 into ZIF-8 to create 6-AF@BIBR 1532@ZIF-8.

They then compared:

1. Free 6-AF@BIBR 1532

2. 6-AF@BIBR 1532@ZIF-8

inside A549 cells, looking at:

• Localization in lysosomes (using LysoTracker)

• Movement into the cytoplasm and nucleus

• Evolution over time (1, 3, 6, 12, 24 hours)

Early Stages (Lysosomal Trapping vs. Escape)

At 1 hour:

• Both free BIBR 1532 and ZIF-8-encapsulated BIBR 1532 appear in lysosomes (yellow signal = green drug + red lysosome).

At 3 hours:

• Lysosomal signal decreases for both.

• For free BIBR 1532, the decreased lysosomal fluorescence is not accompanied by clearly increased signal in cytoplasm or nucleus; much of it seems to be lost or degraded.

• For BIBR 1532@ZIF-8, as lysosomal signal decreases, there is more green fluorescence in the cytoplasm, and some even begins to appear in the nucleus.

At 6 hours and beyond:

• In cells treated with free BIBR 1532, nuclear accumulation is modest and slow.

• In cells treated with BIBR 1532@ZIF-8, nuclear fluorescence becomes much stronger, suggesting more BIBR 1532 reaches the nucleus over time.

Quantitative image analysis confirms that:

• Nuclear fluorescence from BIBR 1532 rises slowly when the drug is free.

• It increases much more steeply and to higher levels when the drug is delivered via ZIF-8.

So encapsulation in ZIF-8 clearly:

• Improves delivery of BIBR 1532 into cells

• Helps it escape the lysosomal trap

• Leads to higher accumulation in the nucleus, where telomerase and hTERT are located

This is a key reason why biological effects are much stronger with BIBR 1532@ZIF-8 than with free BIBR 1532 at the same equivalent dose.

How Does ZIF-8 Encapsulation Change the Biological Effects of BIBR 1532?

The authors compared three conditions in A549 lung cancer cells:

1. ZIF-8 alone

2. BIBR 1532 alone

3. BIBR 1532@ZIF-8 (same effective BIBR 1532 content as (2))

They looked at:

• Cell viability

• hTERT mRNA expression

• Micronucleus formation (a sign of telomere/genome damage)

• Cell cycle distribution

• Cellular senescence

Cell Viability

Using CCK-8 assay:

• BIBR 1532 alone at tested concentrations produced only modest decreases in cell viability after 24 hours.

• ZIF-8 alone did not significantly reduce viability up to 100 µg/mL.

• BIBR 1532@ZIF-8, at concentrations that contain the same amount of BIBR 1532 as the free drug, caused much stronger cytotoxicity.

For example:

• A low concentration of BIBR 1532 alone did not change viability much.

• The equivalent amount of BIBR 1532 delivered via ZIF-8 reduced viability clearly (e.g., about 18% decrease at a low dose, and up to ~80% at the highest tested equivalent dose).

This tells us that better delivery, not just more drug, makes a large difference.

hTERT mRNA Expression

They measured hTERT mRNA levels as a readout of telomerase-related effects.

• BIBR 1532 alone reduced hTERT mRNA in a dose-dependent, but relatively moderate way (e.g., down to around 60–80% of control, depending on dose).

• BIBR 1532@ZIF-8, with the same effective amount of BIBR 1532, reduced hTERT mRNA much more strongly (down to roughly 20% or less of control), almost regardless of the dose tested.

• ZIF-8 alone did not significantly change hTERT mRNA.

So ZIF-8 encapsulation turns BIBR 1532 into a much more potent inhibitor of hTERT expression and telomerase activity within the same time frame.

Micronucleus Formation (Telomere Dysfunction Indicator)

Micronuclei are small, extra nuclei in cells arising from broken or mis-segregated chromosomes. Their presence is a sign of genomic instability and sometimes telomere damage.

• ZIF-8 alone did not increase micronucleus formation.

• Free BIBR 1532 induced some micronucleus formation.

• BIBR 1532@ZIF-8 induced more cells with micronuclei, and more micronuclei per cell.

This indicates that telomere dysfunction and chromosome damage are more pronounced when BIBR 1532 is delivered via ZIF-8.

Cell Cycle Arrest

Telomerase inhibition and telomere dysfunction often lead to cell cycle arrest, especially at the G0/G1 phase, where cells stop preparing for DNA synthesis.

The authors found:

• ZIF-8 alone did not significantly alter the cell cycle.

• Free BIBR 1532 caused slight G0/G1 arrest at the highest dose.

• BIBR 1532@ZIF-8 caused strong G0/G1 arrest, especially at higher doses, where more than 80% of cells could accumulate in G0/G1.

This shows that encapsulated BIBR 1532 is much more effective in forcing cancer cells to stop cycling and dividing.

Cellular Senescence

They also looked at senescence-associated β-galactosidase (SA-β-Gal), a widely used marker of cellular senescence.

• Doxorubicin (DOX) was used as a positive control and induced clear senescence.

• ZIF-8 alone did not induce senescence.

• Free BIBR 1532 induced only a small increase in senescence over 7 days.

• BIBR 1532@ZIF-8 induced strong, dose-dependent senescence, with up to ~89% of cells stained positive at the highest dose.

This fits well with the idea that:

• Telomerase inhibition and telomere dysfunction →

• Cell cycle arrest →

• Long-term growth stop and entry into senescence, rather than immediate apoptosis at these doses.

Why Is This Work Important?

This study is not just about one drug and one carrier. It demonstrates several important general points:

1. Hydrophobic, water-insoluble drugs like BIBR 1532 can have good biological mechanisms but poor practical performance because they simply don’t reach the target efficiently.

2. ZIF-8, as a pH-sensitive, imidazole-containing MOF, can act as a smart nanocarrier that:

   • Improves dispersion and stability of the drug in water

   • Facilitates cellular uptake via endocytosis

   • Uses lysosomal acidity and protonation to trigger release and escape from lysosomes

   • Increases nuclear delivery of the cargo

3. By encapsulating BIBR 1532 in ZIF-8, the authors achieved:

   • Stronger growth inhibition of cancer cells

   • Stronger reduction in hTERT mRNA

   • More micronucleus formation (telomere/genome damage)

   • More pronounced G0/G1 cell cycle arrest

   • Dramatically increased cellular senescence

All of this was obtained without changing the chemical structure of BIBR 1532 itself. The improvement came from nanotechnology and delivery strategy, not from altering the drug.

This suggests that:

• Other water-insoluble small molecule drugs could also benefit from encapsulation in ZIF-8 or similar MOFs.

• MOF-based carriers offer a promising platform for better transport, controlled release, and enhanced intracellular targeting of difficult drugs.

Final Takeaway

In simple terms, the paper shows the following:

• BIBR 1532 is a promising telomerase inhibitor, but its poor water solubility limits its use.

• ZIF-8, a nano-porous, pH-sensitive MOF, was used to encapsulate BIBR 1532 and form a composite called BIBR 1532@ZIF-8.

• This encapsulation:

  • Improved the drug’s stability and dispersion in water

  • Increased cellular uptake

  • Helped the drug escape lysosomes thanks to the protonation of imidazole rings

  • Led to more BIBR 1532 in the nucleus

  • Greatly enhanced its ability to inhibit telomerase, arrest the cell cycle, induce telomere damage, and drive cancer cells into senescence

• ZIF-8 alone did not cause major toxicity or interfere with telomerase, which is important for safety.

Overall, this work provides a clear proof-of-concept that ZIF-8 can be used as an effective drug delivery vehicle for hydrophobic telomerase inhibitors and potentially many other poorly soluble small molecules.