poly(ethylenimine)/DNA complexes following endocytosis is known as the
proton sponge hypothesis, which entails the following steps:

1. PEI/DNA complexes are endocytosed.
2. V-ATPases pump protons into the vesicular interior.
3. Water enters the vesicle to balance the osmotic gradient.
4. The vesicle swells due to the entry of ions and water, leading to rupture.

Which of the following combinations is correct?

1. (A), (B) and (D) only
2. (A), (B) and (C) only
3. (A), (B), (C) and (D)
4. (B), (C) and (D) only

   The correct answer is: (A), (B), (C) and (D) — i.e. option 3.

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   Direct Answer to the Question

   The proton sponge hypothesis describing what happens to poly(ethylenimine) (PEI)/DNA complexes after endocytosis includes all four listed steps: (A), (B), (C) and (D).
   Therefore, the correct option is (A), (B), (C) and (D).

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   Explanation of Each Step (A–D)

   (A) PEI/DNA complexes are endocytosed

   • PEI is a cationic polymer that forms complexes (polyplexes) with negatively charged DNA.​

   • These PEI/DNA complexes bind to the cell surface and are internalized via endocytosis into endosomes.

   ✅ This step is correct and is the initiating event in the proton sponge hypothesis.

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   (B) V-ATPases pump protons into the vesicular interior

   • Inside endosomes, vacuolar-type H⁺-ATPases (V‑ATPases) actively transport protons into the lumen, acidifying the vesicle.

   • PEI has high buffering capacity, so it binds many of these incoming protons, acting as a proton sponge.

   ✅ This step is correct and central to the hypothesis because it drives continuous proton pumping.

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   (C) Water will enter the vesicle to balance the osmotic gradient

   • As more protons are pumped in and buffered by PEI, additional ions (especially Cl⁻) enter to maintain electroneutrality, increasing intravesicular osmolarity.

   • The osmotic gradient draws water into the vesicle, causing it to swell.

   ✅ This step is correct, representing the osmotic consequence of proton and ion accumulation.

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   (D) The vesicle will swell due to the entry of ions and water, leading to rupture

   • Influx of ions and water leads to significant swelling of the endosome.

   • The swelling can cause endosomal membrane rupture, releasing PEI/DNA complexes into the cytoplasm, enabling downstream gene expression.

   ✅ This step is correct and explains how PEI/DNA escapes from endosomes.

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   Why the Other Options Are Incorrect

   Use the above logic to see why:

   • (A), (B) and (D) only – incorrect because it omits the essential osmotic water entry step (C).

   • (A), (B) and (C) only – incorrect because it stops before the crucial vesicle rupture (D) that mediates endosomal escape.

   • (B), (C) and (D) only – incorrect because it ignores the required initial endocytosis of PEI/DNA complexes (A).

   Thus, only (A), (B), (C) and (D) correctly and completely describe the proton sponge hypothesis sequence.